Soluble recombinant botulinum toxin proteins

ABSTRACT

The present invention includes recombinant proteins derived from  Clostridium botulinum  toxins. In particular, soluble recombinant  Clostridium botulinum  type A, type B and type E toxin proteins are provided. Methods which allow for the isolation of recombinant proteins free of significant endotoxin contamination are provided. The soluble, endotoxin-free recombinant proteins are used as immunogens for the production of vaccines and antitoxins. These vaccines and antitoxins are useful in the treatment of humans and other animals at risk of intoxication with clostridial toxin.

This application is a continuation of application Ser. No. 10/271,012,filed Oct. 15, 2002, which was a continuation of application Ser. No.08/704,159, filed Aug. 28, 1996, which was a continuation-in-part ofapplication Ser. No. 08/4-5.496, filed Mar. 16, 1995, now issued as U.S.Pat. No. 5,919,665; the disclosures of each of which applications ishereby incorporated in its entirety by reference herein.

FIELD OF THE INVENTION

The present invention relates to the isolation of polypeptides derivedfrom Clostridium botulinum neurotoxins and the use thereof as immunogensfor the production of vaccines, including multivalent vaccines, andantitoxins.

BACKGROUND OF THE INVENTION

The genus Clostridium is comprised of gram-positive, anaerobic,spore-forming bacilli. The natural habitat of these organisms is theenvironment and the intestinal tracts of humans and other animals.Indeed, clostridia are ubiquitous; they are commonly found in soil,dust, sewage, marine sediments, decaying vegetation, and mud. [See e.g.,P. H. A. Sneath et al., “Clostridium,” Bergey's Manual® of SystematicBacteriology, Vol. 2, pp. 1141-1200, Williams & Wilkins (1986). Despitethe identification of approximately 100 species of Clostridium, only asmall number have been recognized as etiologic agents of medical andveterinary importance. Nonetheless, these species are associated withvery serious diseases, including botulism, tetanus, anaerobiccellulitis, gas gangrene, bacteremia, pseudomembranous colitis, andclostridial gastroenteritis. Table 1 lists some of the species ofmedical and veterinary importance and the diseases with which they areassociated. As virtually all of these species have been isolated fromfecal samples of apparently healthy persons, some of these isolates maybe transient, rather than permanent residents of the colonic flora.TABLE 1 Clostridium Species Of Medical And Veterinary Importance*Species Disease C. aminovalericum Bacteriuria (pregnant women) C.argentinense Infected wounds; Bacteremia; Botulism; Infections ofamniotic fluid C. baratii Infected war wounds; Peritonitis; Infectiousprocesses of the eye, ear and prostate C. beijerinckikii Infected woundsC. bifermentans Infected wounds; Abscesses; Gas Gangrene; Bacteremia C.botulinum Food poisoning; Botulism (wound, food, infant) C. butyricumUrinary tract, lower respiratory tract, pleural cavity, and abdominalinfections; Infected wounds; Abscesses; Bacteremia C. cadaverisAbscesses; Infected wounds C. carnis Soft tissue infections; BacteremiaC. chauvoei Blackleg C. clostridioforme Abdominal, cervical, scrotal,pleural, and other infections; Septicemia; Peritonitis; Appendicitis C.cochlearium Isolated from human disease processes, but role in diseaseunknown. C. difficile Antimicrobial-associated diarrhea;Pseudomembranous enterocolitis; Bacteremia; Pyogenic infections C.fallax Soft tissue infections C. ghnoii Soft tissue infections C.glycolicum Wound infections; Abscesses; Peritonitis C. hastiformeInfected war wounds; Bacteremia; Abscesses C. histolyticum Infected warwounds; Gas gangrene; Gingival plaque isolate C. indolisGastrointestinal tract infections C. innocuum Gastrointestinal tractinfections; empyema C. irregulare Penile lesions C. leptum Isolated fromhuman disease processes, but role in disease unknown. C. limosumBacteremia; Peritonitis; Pulmonary infections C. malenominatum Variousinfectious processes C. novyi Infected wounds; Gas gangrene; Blackleg,Big head (ovine); Redwater disease (bovine) C. oroticum Urinary tractinfections; Rectal abscesses C. paraputrificum Bacteremia; Peritonitis;Infected wounds; Appendicitis C. perfringens Gas gangrene; Anaerobiccellulitis; Intra- abdominal abscesses; Soft tissue infections; Foodpoisoning; Necrotizing pneumonia; Empyema; Meningitis; Bacteremia;Uterine Infections; Enteritis necrotans; Lamb dysentery; Struck; OvineEnterotoxemia; C. putrefaciens Bacteriuria (Pregnant women withbacteremia) C. putrificum Abscesses; Infected wounds; Bacteremia C.ramosum Infections of the abdominal cavity, genital tract, lung, andbiliary tract; Bacteremia C. sartagoforme Isolated from human diseaseprocesses, but role in disease unknown. C. septicum Gas gangrene;Bacteremia; Suppurative infections; Necrotizing enterocolitis; Braxy C.sordellii Gas gangrene; Wound infections; Penile lesions; Bacteremia;Abscesses; Abdominal and vaginal infections C. sphenoides Appendicitis;Bacteremia; Bone and soft tissue infections; Intraperitoneal infections;Infected war wounds; Visceral gas gangrene; Renal abscesses C.sporogenes Gas gangrene; Bacteremia; Endocarditis; central nervoussystem and pleuropulmonary infections; Penile lesions; Infected warwounds; Other pyogenic infections C. subterminale Bacteremia; Empyema;Biliary tract, soft tissue and bone infections C. symbiosum Liverabscesses; Bacteremia; Infections resulting due to bowel flora C.tertium Gas gangrene; Appendicitis; Brain abscesses; Intestinal tractand soft tissue infections; Infected war wounds; Periodontitis;Bacteremia C. tetani Tetanus; Infected gums and teeth; Cornealulcerations; Mastoid and middle ear infections; Intraperitonealinfections; Tetanus neonatorum; Postpartum uterine infections; Softtissue infections, especially related to trauma (including abrasions andlacerations); Infections related to use of contaminated needles C.thermosaccharolyticum Isolated from human disease processes, but role indisease unknown.*Compiled from P. G. Engelkirk et al. “Classification”, Principles andPractice of Clinical Anaerobic Bacteriology, pp. 22-23, Star PublishingCo., Belmont, CA (1992); J. Stephen and R. A. Petrowski, “Toxins WhichTraverse Membranes and Deregulate Cells,” in Bacterial Toxins, 2d ed.,pp. 66-67, American Society for Microbiology (1986); R. Berkow and A. J.Fletcher (eds.), “Bacterial Diseases,” Merck Manual of Diagnosis andTherapy, 16th ed., pp. 116-126,# Merck Research Laboratories, Rahway, N. J. (1992); and O. H. Sigmundand C. M. Fraser (eds.), “Clostridial Infections”, Merck VeterinaryManual, 5th ed., pp. 396-409, Merck & Co., Rahway, N. J. (1979).In most cases, the pathogenicity of these organisms is related to therelease of powerful exotoxins or highly destructive enzymes. Indeed,several species of the genus Clostridium produce toxins and otherenzymes of great medical and veterinary significance. [C. L. Hatheway,Clin. Microbiol. Rev. 3:66-98 (1990).]

Perhaps because of their significance for human and veterinary medicine,much research has been conducted on these toxins, in particular those ofC. botulinum and C. difficile.

C. botulinum

Several strains of Clostridium botulinum produce toxins of significanceto human and animal health. [C. L. Hatheway, Clin. Microbiol. Rev.3:66-98 (1990)] The effects of these toxins range from diarrhealdiseases that can cause destruction of the colon, to paralytic effectsthat can cause death. Particularly at risk for developing clostridialdiseases are neonates and humans and animals in poor health (e.g., thosesuffering from diseases associated with old age or immunodeficiencydiseases).

Clostridium botulinum produces the most poisonous biological toxinknown. The lethal human dose is a mere 10⁻⁹ mg/kg bodyweight for toxinin the bloodstream. Botulinal toxin blocks nerve transmission to themuscles, resulting in flAccId paralysis. When the toxin reaches airwayand respiratory muscles, it results in respiratory failure that cancause death. [S. Arnon, J. Infect. Dis. 154:201-206 (1986)]

C. botulinum spores are carried by dust and are found on vegetablestaken from the soil, on fresh fruits, and on agricultural products suchas honey. Under conditions favorable to the organism, the sporesgerminate to vegetative cells which produces toxin. [S. Amon, Ann. Rev.Med. 31:541 (1980)]

Botulism disease may be grouped into four types, based on the method ofintroduction of toxin into the bloodstream. Food-borne botulism resultsfrom ingesting improperly preserved and inadequately heated food thatcontains botulinal toxin. There were 355 cases of food-borne botulism inthe United States between 1976 and 1984. [K. L. MacDonald et al., Am. J.Epidemiol. 124:794 (1986).] The death rate due to botulinal toxin is 12%and can be higher in particular risk groups. [C. O. Tacket et al., Am.J. Med. 76:794 (1984).] Wound-induced botulism results from C. botulinumpenetrating traumatized tissue and producing toxin that is absorbed intothe bloodstream. Since 1950, thirty cases of wound botulism have beenreported. [M. N. Swartz, “Anaerobic Spore-Forming Bacilli: TheClostridia,” pp. 633-646, in B. D. Davis et al., (eds.), Microbiology,4th edition, J. B. Lippincott Co. (1990).] Inhalation botulism resultswhen the toxin is inhaled. Inhalation botulism has been reported as theresult of accidental exposure in the laboratory [E. Holzer, Med. Klin.41:1735 (1962)] and could arise if the toxin is used as an agent ofbiological warfare [D. R. Franz et al., in Botulinum and TetanusNeurotoxins, B. R. DasGupta, ed., Plenum Press, New York (1993), pp.473-476]. Infectious infant botulism results from C. botulinumcolonization of the infant intestine with production of toxin and itsabsorption into the bloodstream. It is likely that the bacterium gainsentry when spores are ingested and subsequently germinate. [S. Amon, J.Infect. Dis. 154:201 (1986).] There have been 500 cases reported sinceit was first recognized in 1976. [M. N. Swartz, supra.]

Infant botulism strikes infants who are three weeks to eleven months old(greater than 90% of the cases are infants less than six months). [S.Arnon, J. Infect. Dis. 154:201 (1986).] It is believed that infants aresusceptible, due, in large part, to the absence of the full adultcomplement of intestinal microflora. The benign microflora present inthe adult intestine provide an acidic environment that is not favorableto colonization by C. botulinum. Infants begin life with a sterileintestine which is gradually colonized by microflora. Because of thelimited microflora present in early infancy, the intestinal environmentis not as acidic, allowing for C. botulinum spore germination, growth,and toxin production. In this regard, some adults who have undergoneantibiotic therapy which alters intestinal microflora become moresusceptible to botulism.

An additional factor accounting for infant susceptibility to infectiousbotulism is the immaturity of the infant immune system. The matureimmune system is sensitized to bacterial antigens and producesprotective antibodies. Secretory IgA produced in the adult intestine hasthe ability to agglutinate vegetative cells of C. botulinum. [S. Arnon,J. Infect. Dis. 154:201 (1986).] Secretory IgA may also act bypreventing intestinal bacteria and their products from crossing thecells of the intestine. [S. Amon, Epidemiol. Rev. 3:45 (1981).] Theinfant immune system is not primed to do this.

Clinical symptoms of infant botulism range from mild paralysis, tomoderate and severe paralysis requiring hospitalization, to fulminantparalysis, leading to sudden death. [S. Arnon, Epidemiol. Rev. 3:45(1981).]

The chief therapy for severe infant botulism is ventilatory assistanceusing a mechanical respirator and concurrent elimination of toxin andbacteria using cathartics, enemas, and gastric lavage. There were 68hospitalizations in California for infant botulism in a single year witha total cost of over $4 million for treatment. [T. L. Frankovich and S.Arnon, West. J. Med. 154:103 (1991).]

Different strains of Clostridium botulinum each produce antigenicallydistinct toxin designated by the letters A-G. Serotype A toxin has beenimplicated in 26% of the cases of food botulism; types B, E and F havealso been implicated in a smaller percentage of the food botulism cases[H. Sugiyama, Microbiol. Rev. 44:419 (1980)]. Wound botulism has beenreportedly caused by only types A or B toxins [H. Sugiyama, supra].Nearly all cases of infant botulism have been caused by bacteriaproducing either type A or type B toxin. (Exceptionally, one New Mexicocase was caused by Clostridium botulinum producing type F toxin andanother by Clostridium botulinum producing a type B-type F hybrid.) [S.Amon, Epidemiol. Rev. 3:45 (1981).] Type C toxin affects waterfowl,cattle, horses and mink. Type D toxin affects cattle, and type E toxinaffects both humans and birds.

A trivalent antitoxin derived from horse plasma is commerciallyavailable from Connaught Industries Ltd. as a therapy for toxin types A,B, and E. However, the antitoxin has several disadvantages. First,extremely large dosages must be injected intravenously and/orintramuscularly. Second, the antitoxin has serious side effects such asacute anaphylaxis which can lead to death, and serum sickness. Finally,the efficacy of the antitoxin is uncertain and the treatment is costly.[C. O. Tacket et al., Am. J. Med. 76:794 (1984).]

A heptavalent equine botulinal antitoxin which uses only the F(ab′)2portion of the antibody molecule has been tested by the United StatesMilitary. [M. Balady, USAMRDC Newsletter, p. 6 (1991).] This was raisedagainst impure toxoids in those large animals and is not a high titerpreparation.

A pentavalent human antitoxin has been collected from immunized humansubjects for use as a treatment for infant botulism. The supply of thisantitoxin is limited and cannot be expected to meet the needs of allindividuals stricken with botulism disease. In addition, collection ofhuman sera must involve screening out HIV and other potentially serioushuman pathogens. [P. J. Schwarz and S. S. Amon, Western J. Med. 156:197(1992).]

Infant botulism has been implicated as the cause of mortality in somecases of Sudden Infant Death Syndrome (SIDS, also known as crib death).SIDS is officially recognized as infant death that is sudden andunexpected and that remained unexplained despite complete post-mortemexamination. The link of SIDS to infant botulism came when fecal orblood specimens taken at autopsy from SIDS infants were found to containC. botulinum organisms and/or toxin in 3-4% of cases analyzed. [D. R.Peterson et al., Rev. Infect. Dis. 1:630 (1979).] In contrast, only 1 of160 healthy infants (0.6%) had C. botulinum organisms in the feces andno botulinal toxin. (S. Amon et al., Lancet, pp. 1273-76, Jun. 17,1978.)

In developed countries, SIDS is the number one cause of death inchildren between one month and one year old. (S. Arnon et al., Lancet,pp. 1273-77, Jun. 17, 1978.) More children die from SIDS in the firstyear than from any other single cause of death in the first fourteenyears of life. In the United States, there are 8,000-10,000 SIDS victimsannually. Id.

What is needed is an effective therapy against infant botulism that isfree of dangerous side effects, is available in large supply at areasonable price, and can be safely and gently delivered so thatprophylactic application to infants is feasible.

Immunization of subjects with toxin preparations has been done in anattempt to induce immunity against botulinal toxins. A C. botulinumvaccine comprising chemically inactivated (i.e., formaldehyde-treated)type A, B, C, D and E toxin is commercially available for human usage.However, this vaccine preparation has several disadvantages. First, theefficacy of this vaccine is variable (in particular, only 78% ofrecipients produce protective levels of anti-type B antibodies followingadministration of the primary series). Second, immunization is painful(deep subcutaneous inoculation is required for administration), withadverse reactions being common (moderate to severe local reactions occurin approximately 6% of recipients upon initial injection; this numberrises to approximately 11% of individuals who receive boosterinjections) [Informational Brochure for the Pentavalent (ABCDE)Botulinum Toxoid, Centers for Disease Control]. Third, preparation ofthe vaccine is dangerous as active toxin must be handled by laboratoryworkers.

What is needed are safe and effective vaccine preparations foradministration to those at risk of exposure to C. botulinum toxins.

C. difficile

C. difficile, an organism which gained its name due to difficultiesencountered in its isolation, has recently been proven to be anetiologic agent of diarrheal disease. (Sneath et al., p. 1165). C.difficile is present in the gastrointestinal tract of approximately 3%of healthy adults, and 10-30% of neonates without adverse effect(Swartz, at p. 644); by other estimates, C. difficile is a part of thenormal gastrointestinal flora of 2-10% of humans. [G. F. Brooks et al.,(eds.) “Infections Caused by Anaerobic Bacteria,” Jawetz, Melnick, &Adelberg's Medical Microbiology, 19th ed., pp. 257-262, Appleton &Lange, San Mateo, Calif. (1991).] As these organisms are relativelyresistant to most commonly used antimicrobials, when a patient istreated with antibiotics, the other members of the normalgastrointestinal flora are suppressed and C. difficile flourishes,producing cytopathic toxins and enterotoxins. It has been found in 25%of cases of moderate diarrhea resulting from treatment with antibiotics,especially the cephalosporins, clindamycin, and ampicillin. [M. N.Swartz at 644.]

Importantly, C. difficile is commonly associated with nosocomialinfections. The organism is often present in the hospital and nursinghome environments and may be carried on the hands and clothing ofhospital personnel who care for debilitated and immunocompromisedpatients. As many of these patients are being treated withantimicrobials or other chemotherapeutic agents, such transmission of C.difficile represents a significant risk factor for disease. (Engelkirket al., pp. 64-67.) C. difficile is associated with a range ofdiarrhetic illness, ranging from diarrhea alone to marked diarrhea andnecrosis of the gastrointestinal mucosa with the accumulation ofinflammatory cells and fibrin, which forms a pseudomembrane in theaffected area. (Brooks et al.) It has been found in over 95% ofpseudomembranous enterocolitis cases. (Swartz, at p. 644.) Thisoccasionally fatal disease is characterized by diarrhea, multiple smallcolonic plaques, and toxic megacolon. (Swartz, at p. 644.) Althoughstool cultures are sometimes used for diagnosis, diagnosis is best madeby detection of the heat labile toxins present in fecal filtrates frompatients with enterocolitis due to C. difficile. (Swartz, at p. 644-645;and Brooks et al., at p. 260.) C. difficile toxins are cytotoxic fortissue/cell cultures and cause enterocolitis when injected intracecallyinto hamsters. (Swartz, at p. 644.)

The enterotoxicity of C. difficile is primarily due to the action of twotoxins, designated A and B, each of approximately 300,000 in molecularweight. Both are potent cytotoxins, with toxin A possessing directenterocytotoxic activity. [Lyerly et al., Infect. Immun. 60:4633(1992).] Unlike toxin A of C. perfringens, an organism rarely associatedwith antimicrobial-associated diarrhea, the toxin of C. difficile is nota spore coat constituent and is not produced during sporulation.(Swartz, at p. 644.) C. difficile toxin A causes hemorrhage, fluidaccumulation and mucosal damage in rabbit ileal loops and appears toincrease the uptake of toxin B by the intestinal mucosa. Toxin B doesnot cause intestinal fluid accumulation, but it is 1000 times more toxicthan toxin A to tissue culture cells and causes membrane damage.Although both toxins induce similar cellular effects such as actindisaggregation, differences in cell specificity occurs.

Both toxins are important in disease. [Borriello et al., Rev. Infect.Dis., 12(suppl. 2):S185 (1990); Lyerly et al., Infect. Immun., 47:349(1985); and Rolfe, Infect. Immun., 59:1223 (1990).] Toxin A is thoughtto act first by binding to brush border receptors, destroying the outermucosal layer, then allowing toxin B to gain access to the underlyingtissue. These steps in pathogenesis would indicate that the productionof neutralizing antibodies against toxin A may be sufficient in theprophylactic therapy of CDAD. However, antibodies against toxin B may bea necessary additional component for an effective therapeutic againstlater stage colonic disease. Indeed, it has been reported that animalsrequire antibodies to both toxin A and toxin B to be completelyprotected against the disease. [Kim and Rolfe, Abstr. Ann. Meet. Am.Soc. Microbiol., 69:62 (1987).]

C. difficile has also been reported to produce other toxins such as anenterotoxin different from toxins A and B [Banno et al, Rev. Infect.Dis., 6(Suppl. 1:S11-S20 (1984)], a low molecular weight toxin [Rihn etal., Biochem. Biophys. Res. Comm., 124:690-695 (1984)], a motilityaltering factor [Justus et al., Gastroenterol., 83:836-843 (1982)], andperhaps other toxins. Regardless, C. difficile gastrointestinal diseaseis of primary concern.

It is significant that due to its resistance to most commonly usedantimicrobials, C. difficile is associated with antimicrobial therapywith virtually all antimicrobial agents (although most commonlyampicillin, clindamycin and cephalosporins). It is also associated withdisease in patients undergoing chemotherapy with such compounds asmethotrexate, 5-fluorouracil, cyclophosphamide, and doxorubicin. [S. M.Finegold et al., Clinical Guide to Anaerobic Infections, pp. 88-89, StarPublishing Co., Belmont, Calif. (1992).]

Treatment of C. difficile disease is problematic, given the highresistance of the organism. Oral metronidazole, bacitracin andvancomycin have been reported to be effective. (Finegold et al., p. 89.)However there are problems associated with treatment utilizing thesecompounds. Vancomycin is very expensive, some patients are unable totake oral medication, and the relapse rate is high (20-25%), although itmay not occur for several weeks. Id.

C. difficile disease would be prevented or treated by neutralizing theeffects of these toxins in the gastrointestinal tract. Thus, what isneeded is an effective therapy against C. difficile toxin that is freeof dangerous side effects, is available in large supply at a reasonableprice, and can be safely delivered so that prophylactic application topatients at risk of developing pseudomembranous enterocolitis can beeffectively treated.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the reactivity of anti-C. botulinum IgY by Western blot.

FIG. 2 shows the IgY antibody titer to C. botulinum type A toxoid ineggs, measured by ELISA.

FIG. 3 shows the results of C. difficile toxin A neutralization assays.

FIG. 4 shows the results of C. difficile toxin B neutralization assays.

FIG. 5 shows the results of C. difficile toxin B neutralization assays.

FIG. 6 is a restriction map of C. difficile toxin A gene, showingsequences of primers 1-4 (SEQ ID NOS:1-4).

FIG. 7 is a Western blot of C. difficile toxin A reactive protein.

FIG. 8 shows C. difficile toxin A expression constructs.

FIG. 9 shows C. difficile toxin A expression constructs.

FIG. 10 shows the purification of recombinant C. difficile toxin A.

FIG. 11 shows the results of C. difficile toxin A neutralization assayswith antibodies reactive to recombinant toxin A.

FIG. 12 shows the results for a C. difficile toxin A neutralizationplate.

FIG. 13 shows the results for a C. difficile toxin A neutralizationplate.

FIG. 14 shows the results of recombinant C. difficile toxin Aneutralization assays.

FIG. 15 shows C. difficile toxin A expression constructs.

FIG. 16 shows a chromatograph plotting absorbance at 280 nm againstretention time for a pMA1870-680 IgY PEG preparation.

FIG. 17 shows two recombinant C. difficile toxin B expressionconstructs.

FIG. 18 shows C. difficile toxin B expression constructs.

FIG. 19 shows C. difficile toxin B expression constructs.

FIG. 20 shows C. difficile toxin B expression constructs.

FIG. 21 is an SDS-PAGE gel showing the purification of recombinant C.difficile toxin B fusion protein.

FIG. 22 is an SDS-PAGE gel showing the purification of twohistidine-tagged recombinant C. difficile toxin B proteins.

FIG. 23 shows C. difficile toxin B expression constructs.

FIG. 24 is a Western blot of C. difficile toxin B reactive protein.

FIG. 25 shows C. botulinum type A toxin expression constructs;constructs used to provide C. botulinum or C. difficile sequences arealso shown.

FIG. 26 is an SDS-PAGE gel stained with Coomaisse blue showing thepurification of recombinant C. botulinum type A toxin fusion proteins.

FIG. 27 shows C. botulinum type A toxin expression constructs;constructs used to provide C. botulinum sequences are also shown.

FIG. 28 is an SDS-PAGE gel stained with Coomaisse blue showing thepurification of pHisBot protein using the Ni-NTA resin.

FIG. 29 is an SDS-PAGE gel stained with Coomaisse blue showing theexpression of pHisBot protein in BL21(DE3) and BL21(DE3)pLysS hostcells.

FIG. 30 is an SDS-PAGE gel stained with Coomaisse blue showing thepurification of pHisBot protein using a batch absorption procedure.

FIG. 31 is an SDS-PAGE gel stained with Coomaisse blue showing thepurification of pHisBot and pHisBot (native) proteins using a Ni-NTAcolumn.

FIG. 32 is an SDS-PAGE gel stained with Coomaisse blue showing thepurification of pHisBotA protein expressed in pHisBotA(syn) kan lacIqT7/pACYCGro/BL21(DE3) cells using an IDA column.

FIG. 33 is an SDS-PAGE gel stained with Coomaisse blue showing thepurification of pHisBotA, pHisBotB and pHisBotE proteins by IDAchromatography followed by chromatography on S-100 to remove foldingchaperones.

FIG. 34 is an SDS-PAGE gel stained with Coomaisse blue showing theextracts derived from pHisBotB amp T7lac/BL21(DE3) cells before andafter purification on a Ni-NTA column.

FIG. 35 is an SDS-PAGE gel run under native conditions and stained withCoomaisse blue showing the removal of folding chaperones fromIDA-purified BotB protein using a S-100 column.

FIG. 36 is an SDS-PAGE gel stained with Coomaisse blue showing proteinsthat eluted during an imidazole step gradient applied to a IDA columncontaining a lysate of pHisBotB kan lacIq T7/pACYCGro/BL21(DE3) cells.

FIG. 37 is an SDS-PAGE gel run under native conditions and stained withCoomaisse blue showing IDA-purified BotB protein before and afterultrafiltration.

FIG. 38 is an SDS-PAGE gel stained with Coomaisse blue showing thepurification of BotE protein using a NiNTA column.

FIG. 39 is an SDS-PAGE gel stained with Coomaisse blue showing extractsderived from pHisBotA kan T7 lac/BL21(DE3) pLysS cells grown infermentation culture.

FIG. 40 is a chromatogram showing proteins present after IDA-purifiedBotE protein was applied to a S-100 column.

DEFINITIONS

To facilitate understanding of the invention, a number of terms aredefined below.

As used herein, the term “neutralizing” is used in reference toantitoxins, particularly antitoxins comprising antibodies, which havethe ability to prevent the pathological actions of the toxin againstwhich the antitoxin is directed.

As used herein, the term “overproducing” is used in reference to theproduction of clostridial toxin polypeptides in a host cell andindicates that the host cell is producing more of the clostridial toxinby virtue of the introduction of nucleic acid sequences encoding saidclostridial toxin polypeptide than would be expressed by said host cellabsent the introduction of said nucleic acid sequences. To allow ease ofpurification of toxin polypeptides produced in a host cell it ispreferred that the host cell express or overproduce said toxinpolypeptide at a level greater than 1 mg/liter of host cell culture.

“A host cell capable of expressing a recombinant protein at a levelgreater than or equal to 5% of the total cellular protein” is a hostcell in which the recombinant protein represents at least 5% of thetotal cellular protein. To determine what percentage of total cellularprotein the recombinant protein represents, the following steps aretaken. A total of 10 OD₆₀₀ units of recombinant host cells (e.g., 200 μlof cells at OD₆₀₀=50/ml) are removed (at a timepoint known to representthe peak of expression of the desired recombinant protein) to a 1.5 mlmicrofuge tube and pelleted for 2 min at maximum rpm in a microfuge. Thepellets are resuspended in 1 ml of 50 mM NaHPO₄, 0.5 M NaCl, 40 mMimidazole buffer (pH 6.8) containing 1 mg/ml lysozyme. The samples areincubated for 20 min at room temperature and stored ON at −70° C.Samples are thawed completely at room temperature and sonicated 2×10seconds with a Branson Sonifier 450 microtip probe at #3 power setting.The samples are centrifuged for 5 min. at maximum rpm in a microfuge. Analiquot (20 μl) of the protein sample is removed to 20 μl 2× samplebuffer (this represents the total protein extract). The samples areheated to 95° C. for 5 min, then cooled and 5 or 10 μl are loaded onto12.5% SDS-PAGE gels. High molecular weight protein markers are alsoloaded to allow for estimation of the MW of identified recombinantproteins. After electrophoresis, protein is detected generally bystaining with Coomassie blue and the stained gel is scanned using adensitometer to determine the percentage of protein present in eachband. In this manner, the percentage of protein present in the bandcorresponding to the recombinant protein of interest may be determined.It is not necessary that Coomassie blue be employed for the detection ofprotein, a number of fluorescent dyes [e.g., Sypro orange S-6651(Molecular Probes, Eugene, Oreg.] may be employed and the stained gelscanned using a fluoroimager [e.g., Fluor Imager SI (Molecular Dynamics,Sunnyvale, Calif.)].

“A host cell capable of expressing a recombinant protein as a solubleprotein at a level greater than or equal to 0.25% of the total solublecellular protein” is a host cell in which the amount of solublerecombinant protein present represents at least 0.25% of the totalcellular protein. As used herein “total soluble cellular protein” refersto a clarified PEI lysate prepared as described in Example 31(c)(iv).Briefly, cells are harvested following induction of expression ofrecombinant protein (at a point of maximal expression). The cells areresuspended in cell resuspension buffer (CRB: 50 mM NaPO₄, 0.5 M NaCl,40 mM imidazole, pH 6.8) to create a 20% cell suspension (wet weight ofcells/volume of CRB) and cell lysates are prepared as described inExample 31(c)(iv) (i.e., sonication or homogenization followed bycentrifugation). The cell lysate is then flocculated utilizingpolyethyleneimine (PEI) prior to centrifugation. PEI (a 2% solution indH₂O, pH 7.5 with HCl) is added to the cell lysate to a finalconcentration of 0.2%, and stirred for 20 min at room temperature priorto centrifugation [8,500 rpm in JA10 rotor (Beckman) for 30 minutes at4° C.]. This treatment removes RNA, DNA and cell wall components,resulting in a clarified, low viscosity lysate (“PEI clarified lysate”).The recombinant protein present in the PEI clarified lysate is thenpurified (e.g., by chromatography on an IDA column for his-taggedproteins). The amount of purified recombinant protein (i.e., the elutedprotein) is divided by the concentration of protein present in the PEIclarified lysate (typically 8 mg/ml when using a 20% cell suspension asthe starting material) and multiplied by 100 to determine whatpercentage of total soluble cellular protein is comprised of the solublerecombinant protein (see Example 33b).

As used herein, the term “fusion protein” refers to a chimeric proteincontaining the protein of interest (i.e., C. botulinum toxin A, B, C, D,E, F, or G and fragments thereof) joined to an exogenous proteinfragment (the fusion partner which consists of a non-toxin protein). Thefusion partner may enhance solubility of the C. botulinum protein asexpressed in a host cell, may provide an affinity tag to allowpurification of the recombinant fusion protein from the host cell orculture supernatant, or both. If desired, the fusion protein may beremoved from the protein of interest (i.e., toxin protein or fragmentsthereof) prior to immunization by a variety of enzymatic or chemicalmeans known to the art.

As used herein, the term “non-toxin protein” or “non-toxin proteinsequence” refers to that portion of a fusion protein which comprises aprotein or protein sequence which is not derived from a bacterial toxinprotein.

The term “protein of interest”, as used herein, refers to the proteinwhose expression is desired within the fusion protein. In a fusionprotein the protein of interest will be joined or fused with anotherprotein or protein domain, the fusion partner, to allow for enhancedstability of the protein of interest and/or ease of purification of thefusion protein.

As used herein, the term “maltose binding protein” refers to the maltosebinding protein of E. coli. A portion of the maltose binding protein maybe added to a protein of interest to generate a fusion protein; aportion of the maltose binding protein may merely enhance the solubilityof the resulting fusion protein when expressed in a bacterial host. Onthe other hand, a portion of the maltose binding protein may allowaffinity purification of the fusion protein on an amylose resin.

As used herein, the term “poly-histidine tract” when used in referenceto a fusion protein refers to the presence of two to ten histidineresidues at either the amino- or carboxy-terminus of a protein ofinterest. A poly-histidine tract of six to ten residues is preferred.The poly-histidine tract is also defined functionally as being a numberof consecutive histidine residues added to the protein of interest whichallows the affinity purification of the resulting fusion protein on anickel-chelate or IDA column.

As used herein, the term “purified” or “to purify” refers to the removalof contaminants from a sample. For example, antitoxins are purified byremoval of contaminating non-immunoglobulin proteins; they are alsopurified by the removal of immunoglobulin that does not bind toxin. Theremoval of non-immunoglobulin proteins and/or the removal ofimmunoglobulins that do not bind toxin results in an increase in thepercent of toxin-reactive immunoglobulins in the sample. In anotherexample, recombinant toxin polypeptides are expressed in bacterial hostcells and the toxin polypeptides are purified by the removal of hostcell proteins; the percent of recombinant toxin polypeptides is therebyincreased in the sample. Additionally, the recombinant toxinpolypeptides are purified by the removal of host cell components such aslipopolysaccharide (e.g., endotoxin).

The term “recombinant DNA molecule”, as used herein, refers to a DNAmolecule which is comprised of segments of DNA joined together by meansof molecular biological techniques.

The term “recombinant protein” or “recombinant polypeptide”, as usedherein, refers to a protein molecule which is expressed from arecombinant DNA molecule.

The term “native protein”, as used herein, refers to a protein which isisolated from a natural source as opposed to the production of a proteinby recombinant means.

As used, herein the term “portion” when used in reference to a protein(as in “a portion of a given protein”) refers to fragments of thatprotein. The fragments may range in size from four amino acid residuesto the entire amino acid sequence minus one amino acid.

As used herein, the term “soluble” when used in reference to a proteinproduced by recombinant DNA technology in a host cell is a protein whichexists in solution in the cytoplasm of the host cell; if the proteincontains a signal sequence the soluble protein is exported to theperiplasmic space in bacteria hosts and is secreted into the culturemedium in eucaryotic cells capable of secretion or by bacterial hostpossessing the appropriate genes (i.e., the kil gene). In contrast, aninsoluble protein is one which exists in denatured form insidecytoplasmic granules (called inclusion bodies) in the host cell. Highlevel expression (i.e., greater than 10-20 mg recombinant protein/literof bacterial culture) of recombinant proteins often results in theexpressed protein being found in inclusion bodies in the bacterial hostcells. A soluble protein is a protein which is not found in an inclusionbody inside the host cell or is found both in the cytoplasm and ininclusion bodies and in this case the protein may be present at high orlow levels in the cytoplasm.

A distinction is drawn between a soluble protein (i.e., a protein whichwhen expressed in a host cell is produced in a soluble form) and a“solubilized” protein. An insoluble recombinant protein found inside aninclusion body may be solubilized (i.e., rendered into a soluble form)by treating purified inclusion bodies with denaturants such as guanidinehydrochloride, urea or sodium dodecyl sulfate (SDS). These denaturantsmust then be removed from the solubilized protein preparation to allowthe recovered protein to renature (refold). Not all proteins will refoldinto an active conformation after solubilization in a denaturant andremoval of the denaturant. Many proteins precipitate upon removal of thedenaturant. SDS may be used to solubilize inclusion bodies and willmaintain the proteins in solution at low concentration. However,dialysis will not always remove all of the SDS (SDS can form micelleswhich do not dialyze out); therefore, SDS-solubilized inclusion bodyprotein is soluble but not refolded.

A distinction is drawn between proteins which are soluble (i.e.,dissolved) in a solution devoid of significant amounts of ionicdetergents (e.g., SDS) or denaturants (e.g., urea, guanidinehydrochloride) and proteins which exist as a suspension of insolubleprotein molecules dispersed within the solution. A soluble protein willnot be removed from a solution containing the protein by centrifugationusing conditions sufficient to remove bacteria present in a liquidmedium (i.e., centrifugation at 12,000×g for 4-5 minutes). For example,to test whether two proteins, protein A and protein B, are soluble insolution, the two proteins are placed into a solution selected from thegroup consisting of PBS-NaCl (PBS containing 0.5 M NaCl), PBS-NaClcontaining 0.2% Tween 20, PBS, PBS containing 0.2% Tween 20, PBS-C (PBScontaining 2 mM CaCl₂), PBS-C containing either 0.1 or 0.5% Tween 20,PBS-C containing either 0.1 or 0.5% NP-40, PBS-C containing either 0.1or 0.5% Triton X-100, PBS-C containing 0.1% sodium deoxycholate. Themixture containing proteins A and B is then centrifuged at 5000×g for 5minutes. The supernatant and pellet formed by centrifugation are thenassayed for the presence of protein A and B. If protein A is found inthe supernatant and not in the pellet [except for minor amounts (i.e.,less than 10%) as a result of trapping], protein is said to be solublein the solution tested. If the majority of protein B is found in thepellet (i.e., greater than 90%), then protein B is said to exist as asuspension in the solution tested.

As used herein, the term “therapeutic amount” refers to that amount ofantitoxin required to neutralize the pathologic effects of one or moreclostridial toxins in a subject.

The term “pyrogen”, as used herein, refers to a fever-producingsubstance. Pyrogens may be endogenous to the host (e.g., prostaglandins)or may be exogenous compounds (e.g., bacterial endo- and exotoxins,nonbacterial compounds such as antigens and certain steroid compounds,etc.). The presence of pyrogen in a pharmaceutical solution may bedetected using the U.S. Pharmacopeia (USP) rabbit fever test (UnitedStates Pharmacopeia, Vol. XXII (1990) United States PharmacopeialConvention, Rockville, Md., p. 151).

The term “endotoxin”, as used herein, refers to the high molecularweight complexes associated with the outer membrane of grain-negativebacteria. Unpurified endotoxin contains lipids, proteins andcarbohydrates. Highly purified endotoxin does not contain protein and isreferred to as lipopolysaccharide (LPS). Because unpurified endotoxin isof concern in the production of pharmaceutical compounds (e.g., proteinsproduced in E. coli using recombinant DNA technology), the termendotoxin as used herein refers to unpurified endotoxin. Bacterialendotoxin is a well known pyrogen.

As used herein, the term “endotoxin-free” when used in reference to acomposition to be administered parenterally (with the exception ofintrathecal administration) to a host means that the dose to bedelivered contains less than 5 EU/kg body weight [FDA Guidelines forParenteral Drugs (December 1987)]. Assuming a weight of 70 kg for anadult human, the dose must contain less than 350 EU to meet FDAGuidelines for parenteral administration. Endotoxin levels are measuredherein using the Limulus Amebocyte Lysate (LAL) test (Limulus AmebocyteLysate Pyrochrome™, Associates of Cape Cod, Inc. Woods Hole, Mass.). Tomeasure endotoxin levels in preparations of recombinant proteins, 0.5 mlof a solution comprising 0.5 mg of purified recombinant protein in 50 mMNaPO₄, pH 7.0, 0.3M NaCl and 10% glycerol is used in the LAL assayaccording to the manufacturer's instructions for the endpointchromogenic without diazo-coupling method [the specific components ofthe buffer containing recombinant protein to be analyzed in the LAL testare not important; any buffer having a neutral pH may be employed (seefor example, alternative buffers employed in Examples 34, 40 and 45)].Compositions containing less than or equal to 250 endotoxin units(EU)/mg of purified recombinant protein are herein defined as“substantially endotoxin-free.” Preferably the composition contains lessthan or equal to 100, and most preferably less than or equal to 60,(EU)/mg of purified recombinant protein. Typically, administration ofbacterial toxins or toxoids to adult humans for the purpose ofvaccination involves doses of about 10-500 μg protein/dose. Therefore,administration of 10-500 μg of a purified recombinant protein to a 70 kghuman, wherein said purified recombinant protein preparation contains 60EU/mg protein, results in the introduction of only 0.6 to 30 EU (i.e.,0.2 to 8.6% of the maximum allowable endotoxin burden per parenteraldose). Administration of 10-500 μg of a purified recombinant protein toa 70 kg human, wherein said purified recombinant protein preparationcontains 250 EU/mg protein, results in the introduction of only 2.5 to125 EU (i.e., 0.7 to 36% of the maximum allowable endotoxin burden perparenteral dose).

The LAL test is accepted by the U.S. FDA as a means of detectingbacterial endotoxins (21 C.F.R. §§ 660.100-105). Studies have shown thatthe LAL test is equivalent or superior to the USP rabbit pyrogen testfor the detection of endotoxin and thus the LAL test can be used as asurrogate for pyrogenicity studies in animals [F. C. Perason, Pyrogens:endotoxins, LAL testing and depyrogenation, Marcel Dekker, New York(1985), pp. 150-155]. The FDA Bureau of Biologics accepts the LAL assayin place of the USP rabbit pyrogen test so long as the LAL assayutilized is shown to be as sensitive as, or more sensitive as the rabbittest [Fed. Reg., 38, 26130 (1980)].

The term “monovalent” when used in reference to a clostridial vaccinerefers to a vaccine which is capable of provoking an immune response ina host animal directed against a single type of clostridial toxin. Forexample, if immunization of a host with C. botulinum type A toxinvaccine induces antibodies in the immunized host which protect against achallenge with type A toxin but not against challenge with type B, C, D,E, F or G toxins, then the type A vaccine is said to be monovalent. Incontrast, a “multivalent” vaccine provokes an immune response in a hostanimal directed against several (i.e., more than one) clostridialtoxins. For example, if immunization of a host with a vaccine comprisingC. botulinum type A and B toxins induces the production of antibodieswhich protect the host against a challenge with both type A and B toxin,the vaccine is said to be multivalent (in particular, this hypotheticalvaccine is bivalent).

As used herein, the term “immunogenically-effective amount” refers tothat amount of an immunogen required to invoke the production ofprotective levels of antibodies in a host upon vaccination.

The term “protective level”, when used in reference to the level ofantibodies induced upon immunization of the host with an immunogen whichcomprises a bacterial toxin, means a level of circulating antibodiessufficient to protect the host from challenge with a lethal dose of thetoxin.

As used herein, the terms “protein” and “polypeptide” refer to compoundscomprising amino acids joined via peptide bonds and are usedinterchangeably.

The terms “toxin” and “neurotoxin” when used in reference to toxinsproduced by members (i.e., species and strains) of the genus Clostridiumare used interchangeably and refer to the proteins which are poisonousto nerve tissue.

The term “receptor-binding domain” when used in reference to a C.botulinum toxin refers to the carboxy-terminal portion of the heavychain (H_(C) or the C fragment) of the toxin which is presumed to beresponsible for the binding of the active toxin (i.e., the derivativetoxin comprising the H and L chains joined via disulfide bonds) toreceptors on the surface of synaptosomes. The receptor-binding domainfor C. botulinum type A toxin is defined herein as comprising amino acidresidues 861 through 1296 of SEQ ID NO:28. The receptor-binding domainfor C. botulinum type B toxin is defined herein as comprising amino acidresidues 848 through 1291 of SEQ ID NO:40 (strain Eklund 17B). Thereceptor-binding domain of C. botulinum type C1 toxin is defined hereinas comprising amino acid residues 856 through 1291 of SEQ ID NO:60. Thereceptor-binding domain of C. botulinum type D toxin is defined hereinas comprising amino acid residues 852 through 1276 of SEQ ID NO:66. Thereceptor-binding domain of C. botulinum type E toxin is defined hereinas comprising amino acid residues 835 through 1250 of SEQ ID NO:50(Beluga strain). The receptor-binding domain of C. botulinum type Ftoxin is defined herein as comprising amino acid residues 853 through1274 of SEQ ID NO:71. The receptor-binding domain of C. botulinum type Gtoxin is defined herein as comprising amino acid residues 853 through1297 of SEQ ID NO:77. Within a given serotype, small variations in theprimary amino acid sequence of the botulinal toxins isolated fromdifferent strains has been reported [Whelan et al. (1992), supra andMinton (1995) Curr. Top. Microbiol. Immunol. 195:161-194]. The presentinvention contemplates fusion proteins comprising the receptor-bindingdomain of C. botulinum toxins from serotypes A-G including the variantsfound among different strains within a given serotype. Thereceptor-binding domains listed above are used as the prototype for eachstrain within a serotype. Fusion proteins containing an analogous regionfrom a strain other than the prototype strain are encompassed by thepresent invention.

Fusion proteins comprising the receptor binding domain (i.e., Cfragment) of botulinal toxins may include amino acid residues locatedbeyond the termini of the domains defined above. For example, thepHisBotB protein contains amino acid residues 846-1291 of SEQ ID NO:40;this fusion protein thus comprises the receptor-binding domain for C.botulinum type B toxin as defined above (i.e., Ile-848 throughGlu-1291). Similarly, pHisBotE contains amino acid residues 827-1252 ofSEQ ID NO:50 and pHisBotG contains amino acid residues 851-1297 of SEQID NO:77. Thus, both pHisBotE and pHisBotG fusion proteins contain a fewamino acids located beyond the N-terminus of the definedreceptor-binding domain.

The terms “native gene” or “native gene sequences” are used to indicateDNA sequences encoding a particular gene which contain the same DNAsequences as found in the gene as isolated from nature. In contrast,“synthetic gene sequences” are DNA sequences which are used to replacethe naturally occurring DNA sequences when the naturally occurringsequences cause expression problems in a given host cell. For example,naturally-occurring DNA sequences encoding codons which are rarely usedin a host cell may be replaced (e.g., by site-directed mutagenesis) suchthat the synthetic DNA sequence represents a more frequently used codon.The native DNA sequence and the synthetic DNA sequence will preferablyencode the same amino acid sequence.

SUMMARY OF THE INVENTION

The present invention relates to the production of polypeptides derivedfrom toxins particularly in recombinant host cells. In one embodiment,the present invention provides a host cell containing a recombinantexpression vector, said vector encoding a protein comprising at least aportion of a Clostridium botulinum toxin, said toxin selected from thegroup consisting of type B toxin and type E toxin. The present inventionis not limited by the nature of sequences encoding portions of the C.botulinum toxin. These sequences may be derived from the native genesequences or alternatively they may comprise synthetic gene sequences.Synthetic gene sequences are employed when expression of the native genesequences is problematic in a given host cell (e.g., when the nativegene sequences contain sequences resembling yeast transcriptiontermination signals and the desired host cell is a yeast cell).

In one embodiment, the host cell is capable of expressing therecombinant C. botulinum toxin protein at a level greater than or equalto 2% to 40% of the total cellular protein and preferably at a levelgreater than or equal to 5% of the total cellular protein. In anotherembodiment, the host cell is capable of expressing the recombinant C.botulinum toxin protein as a soluble protein at a level greater than orequal to 0.25% of the total cellular protein and preferably at a levelgreater than or equal to 0.25% to 10% of the total cellular protein.

The present invention is not limited by the nature of the host cellemployed for the production of recombinant C. botulinum toxin proteins.In a preferred embodiment, the host cell is an E. coli cell. In anotherpreferred embodiment, the host cell is an insect cell; particularlypreferred insect host cells are Spodoptera frugiperda (Sf9) cells. Inanother preferred embodiment, the host cell is a yeast cell;particularly preferred yeast cells are Pichia pastoris cells.

In another embodiment, the invention provides a host cell containing arecombinant expression vector, said vector encoding a fusion proteincomprising a non-toxin protein sequence and at least a portion of aClostridium botulinum toxin, said toxin selected from the groupconsisting of type B toxin and type E toxin. The invention is notlimited by the nature of the portion of the Clostridium botulinum toxinselected. In a preferred embodiment, the portion of the toxin comprisesthe receptor binding domain (i.e., the C fragment of the toxin). Thepresent invention is not limited by the nature of the non-toxin proteinsequence employed. In a preferred embodiment, the non-toxin proteinsequence comprises a poly-histidine tract. A number of alternativefusion tags or fusion partners are known to the art (e.g., MBP, GST,protein A, etc.) and may be employed for the production of fusionproteins comprising a portion of a botulinal toxin.

The present invention further provides a vaccine comprising a fusionprotein, said fusion protein comprising a non-toxin protein sequence andat least a portion of a Clostridium botulinum toxin, said toxin selectedfrom the group consisting of type B toxin and type E toxin. The vaccinemay be a monovalent vaccine (i.e., containing only a toxin B fusionprotein or a toxin E fusion protein), a bivalent vaccine (i.e.,containing both a toxin B fusion protein and a toxin E fusion protein)or a trivalent or higher valency vaccine. In a preferred embodiment, thetoxin B fusion protein and/or toxin E fusion protein is combined with afusion protein comprising a non-toxin protein sequence and at least aportion of Clostridium botulinum type A toxin. The present invention isnot limited by the nature of the portion of the Clostridium botulinumtoxin selected. In a preferred embodiment, the portion of the toxincomprises the receptor binding domain (i.e., the C fragment of thetoxin). The present invention is not limited by the nature of thenon-toxin protein sequence employed. In a preferred embodiment, thenon-toxin protein sequence comprises a poly-histidine tract. A number ofalternative fusion tags or fusion partners are known to the art (e.g.,MBP, GST, protein A, etc.) and may be employed for the generation offusion proteins comprising vaccines. When a fusion partner (i.e., thenon-toxin protein sequence) is employed for the production of arecombinant C botulinal toxin protein, the fusion partner may be removedfrom the recombinant C botulinal toxin protein if desired (i.e., priorto administration of the protein to a subject) using a variety ofmethods known to the art (e.g., digestion of fusion proteins containingFactorXa or thrombin recognition sites with the appropriate enzyme). Anumber of the pETHis vectors employed herein provide an N-terminalhis-tag followed by a FactorXa cleavage site (see Example 28a); thebotulinal C fragment sequences follow the FactorXa site and thus,FactorXa can be used to remove the his-tag from the botulinal fusionprotein. In a preferred embodiment, the vaccine is substantiallyendotoxin-free.

The present invention is not limited by the method employed for thegeneration of vaccine comprising fusion proteins comprising a non-toxinprotein sequence and at least a portion of a Clostridium botulinumtoxin. The fusion proteins may be produced by recombinant DNA meansusing either native or synthetic gene sequences expressed in a hostcell. The present invention is not limited to the production of vaccinesusing recombinant host cells; cell free in vitrotranscription/translation systems may be employed for the expression ofthe nucleic acid constructs encoding the fusion proteins of the presentinvention. An example of such a cell-free system is the commerciallyavailable TnT™ Coupled Reticulocyte Lysate System (Promega Corporation,Madison, Wis.). Alternatively, the fusion proteins of the presentinvention may be generated by synthetic means (i.e., peptide synthesis).

The present invention further provides a method of generating antibodydirected against a Clostridium botulinum toxin comprising: a) providingin any order: i) an antigen comprising a fusion protein comprising anon-toxin protein sequence and at least a portion of a Clostridiumbotulinum toxin, said toxin selected from the group consisting of type Btoxin and type E toxin, and ii) a host; and b) immunizing the host withthe antigen so as to generate an antibody. In a preferred embodiment,the antigen used to immunize the host also contains a fusion proteincomprising a non-toxin protein sequence and at least a portion ofClostridium botulinum type A toxin. The present invention is not limitedby the nature of the portion of the Clostridium botulinum toxinselected. In a preferred embodiment, the portion of the toxin comprisesthe receptor binding domain (i.e., the C fragment of the toxin). Thepresent invention is not limited by the nature of the non-toxin proteinsequence employed. In a preferred embodiment, the non-toxin proteinsequence comprises a poly-histidine tract. A number of alternativefusion tags or fusion partners are known to the art (e.g., MBP, GST,protein A, etc.) and may be employed for the generation of fusionproteins comprising vaccines. When a fusion partner (i.e., the non-toxinprotein sequence) is employed for the production of a recombinant Cbotulinal toxin protein, the fusion partner may be removed from therecombinant C botulinal toxin protein if desired (i.e., prior toadministration of the protein to a subject) using a variety of methodsknown to the art (e.g., digestion of fusion proteins containing FactorXaor thrombin recognition sites with the appropriate enzyme).

The present invention is not limited by the nature of the host employedfor the production of the antibodies of the invention. In a preferredembodiment, the host is a mammal, preferably a human. The antibodies ofthe present invention may be generated using non-mammalian hosts such asbirds, preferably chickens. In a preferred embodiment the method of thepresent invention further comprised the step c) of collecting theantibodies from the host. In yet another embodiment, the method of thepresent invention further comprises the step d) of purifying theantibodies.

The present invention further provides antibodies raised according tothe above methods.

The present invention further contemplates multivalent vaccinescomprising at least two recombinant C. botulinum toxin proteins derivedfrom the group consisting of C. botulinum serotypes A, B, C, D, E, F,and G. The invention contemplates bivalent, trivalent, quadravalent,pentavalent, heptavalent and septivalent vaccines comprising recombinantC. botulinum toxin proteins. Preferably the recombinant C. botulinumtoxin protein comprises the receptor binding domain (i.e., C fragment)of the toxin.

DESCRIPTION OF THE INVENTION

The present invention contemplates vaccinating humans and other animalswith polypeptides derived from C. botulinum neurotoxins which aresubstantially endotoxin-free. These botulinal peptides are also usefulfor the production of antitoxin. Anti-botulinal toxin antitoxin isuseful for the treatment of patients effected by or at risk of symptomsdue to the action of C. botulinum toxins. The organisms, toxins andindividual steps of the present invention are described separatelybelow.

I. Clostridium Species, Clostridial Diseases and Associated Toxins

A preferred embodiment of the method of the present invention isdirected toward obtaining antibodies against Clostridium species, theirtoxins, enzymes or other metabolic by-products, cell wall components, orsynthetic or recombinant versions of any of these compounds. It iscontemplated that these antibodies will be produced by immunization ofhumans or other animals. It is not intended that the present inventionbe limited to any particular toxin or any species of organism. In oneembodiment, toxins from all Clostridium species are contemplated asimmunogens. Examples of these toxins include the neuramimidase toxin ofC. butyricum, C. sordellii toxins HT and LT, toxins A, B, C, D, E, F,and G of C. botulinum and the numerous C. perfringens toxins. In onepreferred embodiment, toxins A, B, and E of C. botulinum arecontemplated as immunogens. Table 2 below lists various Clostridiumspecies, their toxins and some antigens associated with disease. TABLE 2Clostridial Toxins Toxins and Disease-Associated Organism Antigens C.botulinum A, B, C₁, C₂, D, E, F, G C. butyricum Neuraminidase C.difficile A, B, Enterotoxin (not A nor B), Motility Altering Factor, LowMolecular Weight Toxin, Others C. perfringens α, β, ε, I, γ, δ, ν, φ, κ,λ, μ, υ C. sordelli/C. bifermentans HT, LT, α, β, γ C. novyi α, β, γ, δ,ε, ζ, ν, φ C. septicum α, β, γ, δ C. histolyticum α, β, γ, δ, ε, plusadditional enzymes C. chauvoei α, β, γ, δ

It is not intended that antibodies produced against one toxin will onlybe used against that toxin. It is contemplated that antibodies directedagainst one toxin (e.g., C. perfringens type A enterotoxin) may be usedas an effective therapeutic against one or more toxin(s) produced byother members of the genus Clostridium or other toxin producingorganisms (e.g., Bacillus cereus, Staphylococcus aureus, Streptococcusmutans, Acinetobacter calcoaceticus, Pseudomonas aeruginosa, otherPseudomonas species, etc.). It is further contemplated that antibodiesdirected against the portion of the toxin which binds to mammalianmembranes (e.g., C. perfringens enterotoxin A) can also be used againstother organisms. It is contemplated that these membrane binding domainsare produced synthetically and used as immunogens.

II. Obtaining Antibodies in Non-Mammals

A preferred embodiment of the method of the present invention forobtaining antibodies involves immunization. However, it is alsocontemplated that antibodies could be obtained from non-mammals withoutimmunization. In the case where no immunization is contemplated, thepresent invention may use non-mammals with preexisting antibodies totoxins as well as non-mammals that have antibodies to whole organisms byvirtue of reactions with the administered antigen. An example of thelatter involves immunization with synthetic peptides or recombinantproteins sharing epitopes with whole organism components.

In a preferred embodiment, the method of the present inventioncontemplates immunizing non-mammals with bacterial toxin(s). It is notintended that the present invention be limited to any particular toxin.In one embodiment, toxin from all clostridial bacteria sources (seeTable 2) are contemplated as immunogens. Examples of these toxins are C.butyricum neuramimidase toxin, toxins A, B, C, D, E, F, and G from C.botulinum, C. perfringens toxins α, β, ε, and ι, and C. sordellii toxinsHT and LT. In a preferred embodiment, C. botulinum toxins A, B, C, D, E,and F (or fragments thereof) are contemplated as immunogens.

A particularly preferred embodiment involves the use of bacterial toxinprotein or fragments of toxin proteins produced by molecular biologicalmeans (i.e., recombinant toxin proteins). In a preferred embodiment, theimmunogen comprises the receptor-binding domain (i.e., the ˜50 kDcarboxy-terminal portion of the heavy chain; also referred to as the Cfragment) of C. botulinum serotype A neurotoxin produced by recombinantDNA technology. In another preferred embodiment, the immunogen comprisesthe receptor-binding domain of C. botulinum serotype B neurotoxinproduced by recombinant DNA technology. In yet another preferredembodiment, the immunogen comprises the receptor-binding domain regionof C. botulinum serotype E neurotoxin produced by recombinant DNAtechnology. In yet another preferred embodiment, the immunogen comprisesthe receptor-binding domain region of C. botulinum serotype C1neurotoxin produced by recombinant DNA technology. In yet anotherpreferred embodiment, the immunogen comprises the receptor-bindingdomain region of C. botulinum serotype C2 neurotoxin produced byrecombinant DNA technology. In yet another preferred embodiment, theimmunogen comprises the receptor-binding domain region of C. botulinumserotype D neurotoxin produced by recombinant DNA technology. In yetanother preferred embodiment, the immunogen comprises thereceptor-binding domain region of C. botulinum serotype F neurotoxinproduced by recombinant DNA technology. In yet another preferredembodiment, the immunogen comprises the receptor-binding domain regionof C. botulinum serotype G neurotoxin produced by recombinant DNAtechnology. In a preferred embodiment, the recombinant botulinal toxinproteins are expressed as fusion proteins (e.g., as histidine-taggedproteins). In a still further preferred embodiment, the immunogen is amultivalent vaccine comprising the receptor-binding domain region of C.botulinum toxin from two or more toxins selected from the groupconsisting of type A, type B, type C (including C1 and C2), type D, typeE, and type F toxin.

When immunization is used, the preferred non-mammal is from the classAves. All birds are contemplated (e.g., duck, ostrich, emu, turkey,etc.). A preferred bird is a chicken. Importantly, chicken antibody doesnot fix mammalian complement. [See H. N. Benson et al., J. Immunol.87:616 (1961).] Thus, chicken antibody will normally not cause acomplement-dependent reaction. [A. A. Benedict and K. Yamaga,“Immunoglobulins and Antibody Production in Avian Species,” inComparative Immunology (J. J. Marchaloni, ed.), pp. 335-375, Blackwell,Oxford (1966).] Thus, the preferred antitoxins of the present inventionwill not exhibit complement-related side effects observed withantitoxins known presently.

When birds are used, it is contemplated that the antibody will beobtained from either the bird serum or the egg. A preferred embodimentinvolves collection of the antibody from the egg. Laying hens transportimmunoglobulin to the egg yolk (“IgY”) in concentrations equal to orexceeding that found in serum. [See R. Patterson et al., J. Immunol.89:272 (1962); and S. B. Carroll and B. D. Stollar, J. Biol. Chem.258:24 (1983).] In addition, the large volume of egg yolk producedvastly exceeds the volume of serum that can be safely obtained from thebird over any given time period. Finally, the antibody from eggs ispurer and more homogeneous; there is far less non-immunoglobulin protein(as compared to serum) and only one class of immunoglobulin istransported to the yolk.

When considering immunization with toxins, one may consider modificationof the toxins to reduce the toxicity. In this regard, it is not intendedthat the present invention be limited by immunization with modifiedtoxin. Unmodified (“native”) toxin is also contemplated as an immunogen.

It is also not intended that the present invention be limited by thetype of modification—if modification is used. The present inventioncontemplates all types of toxin modification, including chemical andheat treatment of the toxin. The preferred modification, however, isformaldehyde treatment.

It is not intended that the present invention be limited to a particularmode of immunization; the present invention contemplates all modes ofimmunization, including subcutaneous, intramuscular, intraperitoneal,and intravenous or intravascular injection, as well as per osadministration of immunogen.

The present invention further contemplates immunization with or withoutadjuvant. (Adjuvant is defined as a substance known to increase theimmune response to other antigens when administered with otherantigens.) If adjuvant is used, it is not intended that the presentinvention be limited to any particular type of adjuvant—or that the sameadjuvant, once used, be used all the time. While the present inventioncontemplates all types of adjuvant, whether used separately or incombinations, the preferred use of adjuvant is the use of CompleteFreund's Adjuvant followed sometime later with Incomplete Freund'sAdjuvant. Another preferred use of adjuvant is the use of GerbuAdjuvant. The invention also contemplates the use of RIBI fowl adjuvantand Quil A adjuvant.

When immunization is used, the present invention contemplates a widevariety of immunization schedules. In one embodiment, a chicken isadministered toxin(s) on day zero and subsequently receives toxin(s) inintervals thereafter. It is not intended that the present invention belimited by the particular intervals or doses. Similarly, it is notintended that the present invention be limited to any particularschedule for collecting antibody. The preferred collection time issometime after day 100.

Where birds are used and collection of antibody is performed bycollecting eggs, the eggs may be stored prior to processing forantibody. It is preferred that eggs be stored at 4° C. for less than oneyear.

It is contemplated that chicken antibody produced in this manner can bebuffer-extracted and used analytically. While unpurified, thispreparation can serve as a reference for activity of the antibody priorto further manipulations (e.g., immunoaffinity purification).

III. Increasing the Effectiveness of Antibodies

When purification is used, the present invention contemplates purifyingto increase the effectiveness of both non-mammalian antitoxins andmammalian antitoxins. Specifically, the present invention contemplatesincreasing the percent of toxin-reactive immunoglobulin. The preferredpurification approach for avian antibody is polyethylene glycol (PEG)separation.

The present invention contemplates that avian antibody be initiallypurified using simple, inexpensive procedures. In one embodiment,chicken antibody from eggs is purified by extraction and precipitationwith PEG. PEG purification exploits the differential solubility oflipids (which are abundant in egg yolks) and yolk proteins in highconcentrations of PEG 8000. [Polson et al., Immunol. Comm. 9:495(1980).] The technique is rapid, simple, and relatively inexpensive andyields an immunoglobulin fraction that is significantly purer in termsof contaminating non-immunoglobulin proteins than the comparableammonium sulfate fractions of mammalian sera and horse antibodies. Themajority of the PEG is removed from the precipitated chickenimmunoglobulin by treatment with ethanol. Indeed, PEG-purified antibodyis sufficiently pure that the present invention contemplates the use ofPEG-purified antitoxins in the passive immunization of intoxicatedhumans and animals.

IV. Treatment

The present invention contemplates antitoxin therapy for humans andother animals intoxicated by bacterial toxins. A preferred method oftreatment is by intravenous administration of anti-boutlinal antitoxin;oral administration is also contemplated for other clostridialantitoxins.

A. Dosage of Antitoxin

It was noted by way of background that a balance must be struck whenadministering currently available antitoxin which is usually produced inlarge animals such as horses; sufficient antitoxin must be administeredto neutralize the toxin, but not so much antitoxin as to increase therisk of untoward side effects. These side effects are caused by: i)patient sensitivity to foreign (e.g, horse) proteins; ii) anaphylacticor immunogenic properties of non-immunoglobulin proteins; iii) thecomplement fixing properties of mammalian antibodies; and/or iv) theoverall burden of foreign protein administered. It is extremelydifficult to strike this balance when, as noted above, the degree ofintoxication (and hence the level of antitoxin therapy needed) can onlybe approximated.

The present invention contemplates significantly reducing side effectsso that this balance is more easily achieved. Treatment according to thepresent invention contemplates reducing side effects by usingPEG-purified antitoxin from birds.

In one embodiment, the treatment of the present invention contemplatesthe use of PEG-purified antitoxin from birds. The use of yolk-derived,PEG-purified antibody as antitoxin allows for the administration of: 1)non(mammalian)-complement-fixing, avian antibody; 2) a lessheterogeneous mixture of non-immunoglobulin proteins; and 3) less totalprotein to deliver the equivalent weight of active antibody present incurrently available antitoxins. The non-mammalian source of theantitoxin makes it useful for treating patients who are sensitive tohorse or other mammalian sera.

B. Delivery of Antitoxin

Although it is not intended to limit the route of delivery, the presentinvention contemplates a method for antitoxin treatment of bacterialintoxication in which delivery of antitoxin is oral. In one embodiment,antitoxin is delivered in a solid form (e.g., tablets). In analternative embodiment antitoxin is delivered in an aqueous solution.When an aqueous solution is used, the solution has sufficient ionicstrength to solubilize antibody protein, yet is made palatable for oraladministration. The delivery solution may also be buffered (e.g.,carbonate buffer pH 9.5) which can neutralize stomach acids andstabilize the antibodies when the antibodies are administered orally. Inone embodiment the delivery solution is an aqueous solution. In anotherembodiment the delivery solution is a nutritional formula. Preferably,the delivery solution is infant formula. Yet another embodimentcontemplates the delivery of lyophilized antibody encapsulated ormicroencapsulated inside acid-resistant compounds.

Methods of applying enteric coatings to pharmaceutical compounds arewell known to the art [companies specializing in the coating ofpharmaceutical compounds are available; for example, The Coating Place(Verona, Wis.) and AAI (Wilmington, N.C.)]. Enteric coatings which areresistant to gastric fluid and whose release (i.e., dissolution of thecoating to release the pharmaceutical compound) is pH dependent arecommercially available [for example, the polymethacrylates Eudragit® Land Eudragit® S (Rohm GmbH)]. Eudragit® S is soluble in intestinal fluidfrom pH 7.0; this coating can be used to microencapsulate lyophilizedantitoxin antibodies and the particles are suspended in a solutionhaving a pH above or below pH 7.0 for oral administration. Themicroparticles will remain intact and undissolved until they reached theintestines where the intestinal pH would cause them to dissolve therebyreleasing the antitoxin.

The invention contemplates a method of treatment which can beadministered for treatment of acute intoxication. In one embodiment,antitoxin is administered orally in either a delivery solution or intablet form, in therapeutic dosage, to a subject intoxicated by thebacterial toxin which served as immunogen for the antitoxin.

The invention also contemplates a method of treatment which can beadministered prophylactically. In one embodiment, antitoxin isadministered orally, in a delivery solution, in therapeutic dosage, to asubject, to prevent intoxication of the subject by the bacterial toxinwhich served as immunogen for the production of antitoxin. In anotherembodiment, antitoxin is administered orally in solid form such astablets or as microencapsulated particles. Microencapsulation oflyophilized antibody using compounds such as Eudragit® (Rohm GmbH) orpolyethylene glycol, which dissolve at a wide range of pH units, allowsthe oral administration of solid antitoxin in a liquid form (i.e., asuspension) to recipients unable to tolerate administration of tablets(e.g., children or patients on feeding tubes). In one preferredembodiment the subject is a child. In another embodiment, antibodyraised against whole bacterial organism is administered orally to asubject, in a delivery solution, in therapeutic dosage.

V. Vaccines Against Clostridial Species

The invention contemplates the generation of mono- and multivalentvaccines for the protection of an animal (particularly humans) againstseveral clostridial species. Of particular interest are vaccines whichstimulate the production of a humoral immune response to C. botulinum,C. tetani and C. difficile in humans. The antigens comprising thevaccine preparation may be native or recombinantly produced toxinproteins from the clostridial species listed above. When toxin proteinsare used as immunogens they are generally modified to reduce thetoxicity. This modification may be by chemical or genetic (i.e.,recombinant DNA technology) means. In general genetic detoxification(i.e., the expression of nontoxic fragments in a host cell) is preferredas the expression of nontoxic fragments in a host cell precludes thepresence of intact, active toxin in the final preparation. However, whenchemical modification is desired, the preferred toxin modification isformaldehyde treatment.

The invention contemplates that recombinant C. botulinum toxin proteinsbe used as antigens in mono- and multivalent vaccine preparations.Soluble, substantially endotoxin-free recombinant C. botulinum toxinproteins derived from serotypes A, B and E may be used individually(i.e., as mono-valent vaccines) or in combination (i.e., as amulti-valent vaccine). In addition, the recombinant C. botulinum toxinproteins derived from serotypes A, B and E may be used in conjunctionwith either recombinant or native toxins or toxoids from other serotypesof C. botulinum, C. difficile and C. tetani as antigens for thepreparation of these mono- and multivalent vaccines. It is contemplatedthat, due to the structural similarity of C. botulinum and C. tetanitoxin proteins, a vaccine comprising C. difficile and botulinum toxinproteins (native or recombinant or a mixture thereof) be used tostimulate an immune response against C. botulinum, C. tetani and C.difficile.

The present invention further contemplates multi-valent vaccinescomprising two or more botulinal toxin proteins selected from the groupcomprising recombinant C. botulinum toxin proteins derived fromserotypes A, B, C (including C1 and C2), D, E, F and G.

The adverse consequences of exposure to botulinal toxin would be avoidedby immunization of subjects at risk of exposure to the toxin withnontoxic preparations which confer immunity such as chemically orgenetically detoxified toxin.

Vaccines which confer immunity against one or more of the toxin types A,B, E, F and G would be useful as a means of protecting humans from thedeleterious effects of those C. botulinum toxins known to affect man.Indeed as the possibility exists that humans could be exposed to any ofthe seven serotypes of C. botulinum toxin (e.g., during biologicalwarfare or the production of toxin in a laboratory setting), multivalentvaccines capable of conferring immunity against toxin types A-G(including both C1 and C2 toxins) would be useful for the protection ofhumans. Vaccines which confer immunity against one or more of the toxintypes C, D and E would be useful for veterinary applications.

The botulinal neurotoxin is synthesized as a single polypeptide chainwhich is processed into a heavy (H; ˜100 kD) and a light (L; ˜50 kD)chain by cleavage with proteolytic enzymes; these two chains are heldtogether via disulfide bonds in the active toxin (referred to asderivative toxin) [B. R. DasGupta and H. Sugiyama, Biochem. Biophys.Res. Commun. 48:108 (1972); reviewed in B. R. DasGupta, J. Physiol.84:220 (1990), H. Sugiyama, Microbiol. Rev. 44:419 (1980) and C. L.Hatheway, Clin. Microbiol. Rev. 3:66 (1990)]. The heavy chain of theactive toxin is cleaved by trypsin to produce two fragments termed H_(C)(also referred to as H₁ or C) and H_(N) (also referred to as H₂ or B).The H_(C) fragment (˜46 kD) comprises the carboxy end of the H chain.The H_(N) fragment (˜49 kD) comprises the amino end and remains attachedto the L chain (H_(N)L). Neither H_(C) or H_(N)L is toxic. H_(C)competes with whole derivative toxin for binding to synaptosomes andtherefore H_(C) is said to contain the receptor binding site. The H_(C)and H_(N) fragments of botulinal toxin are analogous to the fragments Cand B of tetanus toxin which are produced by papain cleavage. The Cfragment of tetanus toxin has been shown to be responsible for thebinding of tetanus toxin to purified gangliosides and neuronal cells[Halpern and Loftus, J. Biol. Chem. 288:11188 (1993)].

Antisera raised against purified preparations of isolated botulinal Hand L chains have been shown to protect mice against the lethal effectsof the toxin; however, the effectiveness of the two antisera differ withthe anti-H sera being more potent (H. Sugiyama, supra). While thedifferent botulinal toxins show structural similarity to one another,the different serotypes are reported to be immunologically distinct(i.e., sera raised against one toxin type does not cross-react to asignificant degree with other types). Thus, the generation ofmultivalent vaccines may require the use of more than one type of toxin.

C. botulinum toxin genes from all seven serotypes have been cloned andsequenced (Minton (1995), supra); in addition, partial amino acidsequence is available for a number of C. botulinum toxins isolated fromdifferent strains within a given serotype. The C. botulinum toxinscontain about 1250-1300 amino acid residues. On the DNA level, theoverall degree of homology between C. botulinum serotypes A, B, C, D andE toxins averages between 50 and 60% identity with a greater degree ofhomology being found between H chain-encoding regions than between thoseencoding L chains [Whelan et al. (1992) Appl. Environ. Microbiol.58:2345]. The degree of identity between C. botulinum toxins on theamino acid level reflects the level of DNA sequence homology. The mostdivergent area of DNA and amino acid sequence is found within thecarboxy-terminal area of the various C. botulinum H chain genes. Thisportion of the toxin (i.e., H_(C) or the C fragment) plays a major rolein cell binding. As toxin from different serotypes is thought to bind todistinct cell receptor molecules, it is not surprising that the toxinsdiverge significantly over this region.

Within a given serotype, small variations in the primary amino acidsequence of the botulinal toxins isolated from different strains hasbeen reported [Whelan et al. (1992), supra and Minton (1995), supra].The present invention contemplates fusion proteins comprising portionsof C. botulinum toxins from serotypes A-G including the variants foundamong different strains within a given serotype. The present inventionprovides oligonucleotide primers which may be used to amplify the Cfragment or receptor-binding region of the toxin gene from variousstrains of C. botulinum serotype A, serotype B, serotype C (C1 and C2),serotype D, serotype E, serotype F and serotype G. A large number ofdifferent strains of C. botulinum serotype A, serotype B, serotype C,serotype D serotype E and serotype F are available from the AmericanType Culture Collection (ATCC; Rockville, Md.). For example, the ATCCprovides the following: Type A strains: 174 (ATCC 3502), 457 (ATCC17862), and NCTC 7272 (ATCC 19397); Type B strains: 34 (ATCC 439), 62A(ATCC 7948), NCA 213 B (ATCC 7949), 13114 (ATCC 8083), 3137 (ATCC17780), 1347 (ATCC 17841), 2017 (ATCC 17843), 2217 (ATCC 17844), 2254(ATCC 17845) and VP 1731 (ATCC 25765); Type C strains: 2220 (ATCC17782), 2239 (ATCC 17783), 2223 (ATCC 17784; a type C-β strain; C-βstrains produce C2 toxin), 662 (ATCC 17849; a type C-α strain; C-αstrains produce mainly C1 toxin and a small amount of C2 toxin), 2021(ATCC 17850; a type C-α strain) and VPI 3803 (ATCC 25766); Type Dstrains: ATCC 9633, 2023 (ATCC 17851), and VPI 5995 (ATCC 27517); Type Estrains: ATCC 43181, 36208 (ATCC 9564), 2231 (ATCC 17786), 2229 (ATCC17852), 2279 (ATCC 17854) and 2285 (ATCC 17855) and Type F strains: 202F(ATCC 23387), VPI 4404 (ATCC 25764), VPI 2382 (ATCC 27321) and Langeland(ATCC 35415). Type G strain, 113/30 (NCFB 3012) may be obtained from theNational Collection of Food Bacteria (NCFB, AFRC Institute of FoodResearch, Reading, United Kingdom).

Purification methods have been reported for native toxin types A, B, C,D, E, and F [reviewed in G. Sakaguchi, Pharmac. Ther. 19:165 (1983)]. Asthe different botulinal toxins are structurally related, the inventioncontemplates the expression of any of the botulinal toxins (e.g, typesA-G) as soluble recombinant fusion proteins.

In particular, methods for purification of the type A botulinumneurotoxin have been developed [L. J. Moberg and H. Sugiyama, Appl.Environ. Microbiol. 35:878 (1978)]. Immunization of hens with detoxifiedpurified protein results in the generation of neutralizing antibodies[B. S. Thalley et al., in Botulinum and Tetanus Neurotoxins, B. R.DasGupta, ed., Plenum Press, New York (1993), p. 467].

The currently available C. botulinum pentavalent vaccine comprisingchemically inactivated (i.e., formaldehyde treated) type A, B, C, D andE toxins is not adequate. The efficacy is variable (in particular, only78% of recipients produce protective levels of anti-type B antibodiesfollowing administration of the primary series) and immunization ispainful (deep subcutaneous inoculation is required for administration),with adverse reactions being common (moderate to severe local reactionsoccur in approximately 6% of recipients upon initial injection; thisnumber rises to approximately 11% of individuals who receive boosterinjections) [Informational Brochure for the Pentavalent (ABCDE)Botulinum Toxoid, Centers for Disease Control]. Preparation of thisvaccine is dangerous as active toxin must be handled by laboratoryworkers.

In general, chemical detoxification of bacterial toxins using agentssuch as formaldehyde, glutaraldehyde or hydrogen peroxide is not optimalfor the generation of vaccines or antitoxins. A delicate balance must bestruck between too much and too little chemical modification. If thetreatment is insufficient, the vaccine may retain residual toxicity. Ifthe treatment is too excessive, the vaccine may lose potency due todestruction of native immunogenic determinants. Another major limitationof using botulinal toxoids for the generation of antitoxins or vaccinesis the high production expense. For the above reasons, the developmentof methods for the production of nontoxic but immunogenic C. botulinumtoxin proteins is desirable.

The C. botulinum and C tetanus toxin proteins have similar structures[reviewed in E. J. Schantz and E. A. Johnson, Microbiol. Rev. 56:80(1992)]. The carboxy-terminal 50 kD fragment of the tetanus toxin heavychain (fragment C) is released by papain cleavage and has been shown tobe non-toxic and immunogenic. Recombinant tetanus toxin fragment C hasbeen developed as a candidate vaccine antigen [A. J. Makoff et al.,Bio/Technology 7:1043 (1989)]. Mice immunized with recombinant tetanustoxin fragment C were protected from challenge with lethal doses oftetanus toxin. No studies have demonstrated that the recombinant tetanusfragment C protein confers immunity against other botulinal toxins suchas the C. botulinum toxins.

Recombinant tetanus fragment C has been expressed in E. coli (A. J.Makoff et al., Bio/Technology, supra and Nucleic Acids Res. 17:10191(1989); J. L. Halpern et al., Infect. Immun. 58:1004 (1990)], yeast [M.A. Romanos et al., Nucleic Acids Res. 19:1461 (1991)] and baculovirus[I. G. Charles et al., Infect. Immun. 59:1627 (1991)].

Synthetic tetanus toxin genes had to be constructed to facilitateexpression in yeast (M. A. Romanos et al., supra) and E. coli [A. J.Makoff et al., Nucleic Acids Res., supra], due to the high A-T contentof the tetanus toxin gene sequences. High A-T content is a commonfeature of clostridial genes (M. R. Popoff et al., Infect. Immun.59:3673 (1991); H. F. LaPenotiereu et al., in Botulinum and TetanusNeurotoxins, B. R. DasGupta, ed., Plenum Press, New York (1993), p. 463]which creates expression difficulties in E. coli and yeast due primarilyto altered codon usage frequency and fortuitous polyadenylation sites,respectively.

The C fragment of the C. botulinum type A neurotoxin heavy chain hasbeen evaluated as a vaccine candidate. The C. botulinum type Aneurotoxin gene has been cloned and sequenced [D. E. Thompson et al.,Eur. J. Biochem. 189:73 (1990)]. The C fragment of the type A toxin wasexpressed as either a fusion protein comprising the botulinal C fragmentfused with the maltose binding protein (MBP) or as a native protein [H.F. LaPenotiere et al, (1993) supra, H. F. LaPenotiere et al., Toxicon.33:1383 (1995) and Middlebrook and Brown (1995), Curr. Top. Microbiol.Immunol. 195:89-122]. The plasmid construct encoding the native proteinwas reported to be unstable, while the fusion protein was expressedprimarily in inclusion bodies as insoluble protein. Immunization of micewith crudely purified MBP fusion protein resulted in protection againstIP challenge with 3 LD₅₀ doses of toxin [LaPenotiere et al., (1993) and(1995), supra]. However, this recombinant C. botulinum type A toxin Cfragment/MBP fusion protein is not a suitable immunogen for theproduction of vaccines as it is expressed as an insoluble protein in E.coli. Furthermore, this recombinant C. botulinum type A toxin Cfragment/BP fusion protein was not shown to be substantially free ofendotoxin contamination. Experience with recombinant C. botulinum type Atoxin C fragment/MBP fusion proteins shows that the presence of the MBPon the fusion protein greatly complicates the removal of endotoxin frompreparations of the recombinant fusion protein (see Ex. 24, infra).Expression of a synthetic gene encoding C. botulinum type A toxin Cfragment as a soluble protein excreted from insect cells has beenreported [Middlebrook and Brown (1995), supra]; no details regarding thelevel of expression achieved or the presence of endotoxin or otherpyrogens were provided. Like the insoluble protein expressed in E. coli,immunization with the recombinant protein produced in insect cells wasreported to protect mice from challenge with C. botulinum toxin A.

Inclusion body protein must be solubilized prior to purification and/oradministration to a host. The harsh treatment of inclusion body proteinneeded to accomplish this solubilization may reduce the immunogenicityof the purified protein. Ideally, recombinant proteins to be used asvaccines are expressed as soluble proteins at high levels (i.e., greaterthan or equal to about 0.75% of total cellular protein) in E. coli orother host cells (e.g., yeast, insect cells, etc.). This facilitates theproduction and isolation of sufficient quantities of the immunogen in ahighly purified form (i.e., substantially free of endotoxin or otherpyrogen contamination). The ability to express recombinant toxinproteins as soluble proteins in E. coli is advantageous due to the lowcost of growth compared to insect or mammalian tissue culture cells.

The C. botulinum type B neurotoxin gene has been cloned and sequencedfrom two strains of C. botulinum type B [Whelan et al. (1992) Appl.Environ. Microbiol. 58:2345 (Danish strain) and Hutson et al. (1994)Curr. Microbiol. 28:101 (Eklund 17B strain)]. The nucleotide sequence ofthe toxin gene derived from the Eklund 17B strain (ATCC 25765) isavailable from the EMBL/GemBank sequence data banks under the accessionnumber X71343; the nucleotide sequence of the coding region is listed inSEQ ID NO:39. The amino acid sequence of the C. botulinum type Bneurotoxin derived from the strain Eklund 17B is listed in SEQ ID NO:40.The nucleotide sequence of the C. botulinum serotype B toxin genederived from the Danish strain is listed in SEQ ID NO:41. The amino acidsequence of the C. botulinum type B neurotoxin derived from the Danishstrain is listed in SEQ ID NO:42.

The C. botulinum type B neurotoxin gene is synthesized as a singlepolypeptide chain which is processed to form a dimer composed of a lightand a heavy chain linked via disulfide bonds. The light chain isresponsible for pharmacological activity (i.e., inhibition of therelease of acetylcholine at the neuromuscular junction). The N-terminalportion of the heavy chain is thought to mediate channel formation whilethe C-terminal portion mediates toxin binding; the type B neurotoxin hasbeen reported to exist as a mixture of predominantly single chain withsome double chain (Whelan et al., supra). The 50 kD carboxy-terminalportion of the heavy chain is referred to as the C fragment or the H_(C)domain. The present invention reports for the first time, the expressionof the C fragment of C. botulinum type B toxin in heterologous hosts(e.g., E. coli).

The C. botulinum type E neurotoxin gene has been cloned and sequencedfrom a number of different strains [Poulet et al. (1992) Biochem.Biophys. Res. Commun. 183:107; Whelan et al. (1992) Eur. J. Biochem.204:657; and Fujii et al. (1993) J. Gen. Microbiol. 139:79]. Thenucleotide sequence of the type E toxin gene is available from the EMBLsequence data bank under accession numbers X62089 (strain Beluga) andX62683 (strain NCTC 11219); the nucleotide sequence of the coding region(strain Beluga) is listed in SEQ ID NO:45. The amino acid sequence ofthe C. botulinum type E neurotoxin derived from strain Beluga is listedin SEQ ID NO:46. The type E neurotoxin gene is synthesized as a singlepolypeptide chain which may be converted to a double-chain form (i.e., aheavy chain and a light chain) by cleavage with trypsin; unlike the typeA neurotoxin, the type E neurotoxin exists essentially only in thesingle-chain form. The 50 kD carboxy-terminal portion of the heavy chainis referred to as the C fragment or the H_(C) domain. The presentinvention reports for the first time, the expression of the C fragmentof C. botulinum type E toxin in heterologous hosts (e.g., E. coli).

The C. botulinum type C1, D, F and G neurotoxin genes have been clonedand sequenced. The nucleotide and amino acid sequences of these genesand toxins are provided herein. The invention provides methods for theexpression of the C fragment from each of these toxin genes inheterologous hosts and the purification of the resulting recombinantproteins.

The subject invention provides methods which allow the production ofsoluble C. botulinum toxin proteins in economical host cells (e.g., E.coli). In addition the subject invention provides methods which allowthe production of soluble botulinal toxin proteins in yeast and insectcells. Further, methods for the isolation of purified soluble C.botulinum toxin proteins which are suitable for immunization of humansand other animals are provided. These soluble, purified preparations ofC. botulinum toxin proteins provide the basis for improved vaccinepreparations and facilitate the production of antitoxin.

When recombinant clostridial toxin proteins produced in gram-negativebacteria (e.g., E. coli) are used as vaccines, they are purified toremove endotoxin prior to administration to a host animal. In order tovaccinate a host, an immunogenically-effective amount of purifiedsubstantially endotoxin-free recombinant clostridial toxin protein isadministered in any of a number of physiologically acceptable carriersknown to the art. When administered for the purpose of vaccination, thepurified substantially endotoxin-free recombinant clostridial toxinprotein may be used alone or in conjunction with known adjutants,including potassium alum, aluminum phosphate, aluminum hydroxide, Gerbuadjuvant (GMDP; C.C. Biotech Corp.), RIBI adjuvant (MPL; RIBIImmunochemical Research, Inc.), QS21 (Cambridge Biotech). The alum andaluminum-based adjutants are particularly preferred when vaccines are tobe administered to humans; however, any adjuvant approved for use inhumans may be employed. The route of immunization may be nasal, oral,intramuscular, intraperitoneal or subcutaneous.

The invention contemplates the use of soluble, substantiallyendotoxin-free preparations of fusion proteins comprising the C fragmentof the C. botulinum type A, B, C, D, E, F, and G toxin as vaccines. Inone embodiment, the vaccine comprises the C fragment of either the C.botulinum type A, B, C, D, E, F, or G toxin and a poly-histidine tract(also called a histidine tag). In a particularly preferred embodiment, afusion protein comprising the histidine tagged C fragment is expressedusing the pET series of expression vectors (Novagen). The pET expressionsystem utilizes a vector containing the T7 promoter which encodes thefusion protein and a host cell which can be induced to express the T7DNA polymerase (i.e., a DE3 host strain). The production of C fragmentfusion proteins containing a histidine tract is not limited to the useof a particular expression vector and host strain. Several commerciallyavailable expression vectors and host strains can be used to express theC fragment protein sequences as a fusion protein containing a histidinetract (For example, the pQE series (pQE-8, 12, 16, 17, 18, 30, 31, 32,40, 41, 42, 50, 51, 52, 60 and 70) of expression vectors (Qiagen) whichare used with the host strains M15[pREP4] (Qiagen) and SG13009[pREP4](Qiagen) can be used to express fusion proteins containing six histidineresidues at the amino-terminus of the fusion protein). Furthermore anumber of commercially available expression vectors which provide ahistidine tract also provide a protease cleavage site between thehistidine tract and the protein of interest (e.g., botulinal toxinsequences). Cleavage of the resulting fusion protein with theappropriate protease will remove the histidine tag from the protein ofinterest (e.g., botulinal toxin sequences) (see Example 28a, infra).Removal of the histidine tag may be desirable prior to administration ofthe recombinant botulinal toxin protein to a subject (e.g., a human).

VI. Detection of Toxin

The invention contemplates detecting bacterial toxin in a sample. Theterm “sample” in the present specification and claims is used in itsbroadest sense. On the one hand it is meant to include a specimen orculture. On the other hand, it is meant to include both biological andenvironmental samples.

Biological samples may be animal, including human, fluid, solid (e.g.,stool) or tissue; liquid and solid food products and ingredients such asdairy items, vegetables, meat and meat by-products, and waste.Environmental samples include environmental material such as surfacematter, soil, water and industrial samples, as well as samples obtainedfrom food and dairy processing instruments, apparatus, equipment,disposable and non-disposable items. These examples are not to beconstrued as limiting the sample types applicable to the presentinvention.

The invention contemplates detecting bacterial toxin by a competitiveimmunoassay method that utilizes recombinant toxin A and toxin Bproteins, antibodies raised against recombinant bacterial toxinproteins. A fixed amount of the recombinant toxin proteins areimmobilized to a solid support (e.g., a microtiter plate) followed bythe addition of a biological sample suspected of containing a bacterialtoxin. The biological sample is first mixed with affinity-purified orPEG fractionated antibodies directed against the recombinant toxinprotein. A reporter reagent is then added which is capable of detectingthe presence of antibody bound to the immobilized toxin protein. Thereporter substance may comprise an antibody with binding specificity forthe antitoxin attached to a molecule which is used to identify thepresence of the reporter substance. If toxin is present in the sample,this toxin will compete with the immobilized recombinant toxin proteinfor binding to the anti-recombinant antibody thereby reducing the signalobtained following the addition of the reporter reagent. A control isemployed where the antibody is not mixed with the sample. This gives thehighest (or reference) signal.

The invention also contemplates detecting bacterial toxin by a“sandwich” immunoassay method that utilizes antibodies directed againstrecombinant bacterial toxin proteins. Affinity-purified antibodiesdirected against recombinant bacterial toxin proteins are immobilized toa solid support (e.g., microtiter plates). Biological samples suspectedof containing bacterial toxins are then added followed by a washing stepto remove substantially all unbound antitoxin. The biological sample isnext exposed to the reporter substance, which binds to antitoxin and isthen washed free of substantially all unbound reporter substance. Thereporter substance may comprise an antibody with binding specificity forthe antitoxin attached to a molecule which is used to identify thepresence of the reporter substance. Identification of the reportersubstance in the biological tissue indicates the presence of thebacterial toxin.

It is also contemplated that bacterial toxin be detected by pouringliquids (e.g. soups and other fluid foods and feeds includingnutritional supplements for humans and other animals) over immobilizedantibody which is directed against the bacterial toxin. It iscontemplated that the immobilized antibody will be present in or on suchsupports as cartridges, columns, beads, or any other solid supportmedium. In one embodiment, following the exposure of the liquid to theimmobilized antibody, unbound toxin is substantially removed by washing.The exposure of the liquid is then exposed to a reporter substance whichdetects the presence of bound toxin. In a preferred embodiment thereporter substance is an enzyme, fluorescent dye, or radioactivecompound attached to an antibody which is directed against the toxin(i.e., in a “sandwich” immunoassay). It is also contemplated that thedetection system will be developed as necessary (e.g., the addition ofenzyme substrate in enzyme systems; observation using fluorescent lightfor fluorescent dye systems; and quantitation of radioactivity forradioactive systems).

EXPERIMENTAL

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

In the disclosure which follows, the following abbreviations apply: ° C.(degrees Centigrade); rpm (revolutions per minute); BBS-Tween (boratebuffered saline containing Tween); BSA (bovine serum albumin); ELISA(enzyme-linked immunosorbent assay); CFA (complete Freund's adjuvant);IFA (incomplete Freund's adjuvant); IgG (immunoglobulin G); IgY(immunoglobulin Y); IM (intramuscular); IP (intraperitoneal); IV(intravenous or intravascular); SC (subcutaneous); H₂O (water); HCl(hydrochloric acid); LD₁₀₀ (lethal dose for 100% of experimentalanimals); aa (amino acid); HPLC (high performance liquidchromatography); kD (kilodaltons); gm (grams); μg (micrograms); mg(milligrams); ng (nanograms); μl (microliters); ml (milliliters); mm(millimeters); nm (nanometers); μm (micrometer); M (molar); mM(millimolar); MW (molecular weight); sec (seconds); min(s)(minute/minutes); hr(s) (hour/hours); MgCl, (magnesium chloride); NaCl(sodium chloride); Na₂CO₃ (sodium carbonate); OD₂₈₀ (optical density at280 nm); OD₆₀₀ (optical density at 600 nm); PAGE (polyacrylamide gelelectrophoresis); PBS [phosphate buffered saline (150 mM NaCl, 10 mMsodium phosphate buffer, pH 7.2)]; PEG (polyethylene glycol); PMSF(phenylmethylsulfonyl fluoride); SDS (sodium dodecyl sulfate); Tris(tris(hydroxymethyl)aminomethane); Ensure® ((Ensure®, Ross Laboratories,Columbus Ohio); Enfamil® (Enfamil®, Mead Johnson); w/v (weight tovolume); v/v (volume to volume); Amicon (Amicon, Inc., Beverly, Mass.);Amresco (Amresco, Inc., Solon, Ohio); ATCC (American Type CultureCollection, Rockville, Md.); BBL (Baltimore Biologics Laboratory, (adivision of Becton Dickinson), Cockeysville, Md.); Becton Dickinson(Becton Dickinson Labware, Lincoln Park, N.J.); BioRad (BioRad,Richmond, Calif.); Biotech (C-C Biotech Corp., Poway, Calif.); CharlesRiver (Charles River Laboratories, Wilmington, Mass.); Cocalico(Cocalico Biologicals Inc., Reamstown, Pa.); CytRx (CytRx Corp.,Norcross, Ga.); Falcon (e.g. Baxter Healthcare Corp., McGaw Park, Ill.and Becton Dickinson); FDA (Federal Food and Drug Administration);Fisher Biotech (Fisher Biotech, Springfield, N.J.); GIBCO (Grand IslandBiologic Company/BRL, Grand Island, N.Y.); Gibco-BRL (Life Technologies,Inc., Gaithersburg, Md.); Harlan Sprague Dawley (Harlan Sprague Dawley,Inc., Madison, Wis.); Mallinckrodt (a division of Baxter HealthcareCorp., McGaw Park, Ill.); Millipore (Millipore Corp., Marlborough,Mass.); New England Biolabs (New England Biolabs, Inc., Beverly, Mass.);Novagen (Novagen, Inc., Madison, Wis.); Pharmacia (Pharmacia, Inc.,Piscataway, N.J.); Qiagen (Qiagen, Chatsworth, Calif.); Sasco (Sasco,Omaha, Nebr.); Showdex (Showa Denko America, Inc., New York, N.Y.);Sigma (Sigma Chemical Co., St. Louis, Mo.); Sterogene (Sterogene, Inc.,Arcadia, Calif.); Tech Lab (Tech Lab, Inc., Blacksburg, Va.); andVaxcell (Vaxcell, Inc., a subsidiary of CytRx Corp., Norcross, Ga.).

When a recombinant protein is described in the specification it isreferred to in a short-hand manner by the amino acids in the toxinsequence present in the recombinant protein rounded to the nearest 10.For example, the recombinant protein pMB1850-2360 contains amino acids1852 through 2362 of the C. difficile toxin B protein. The specificationgives detailed construction details for all recombinant proteins suchthat one skilled in the art will know precisely which amino acids arepresent in a given recombinant protein.

Example 1 Production of High-Titer Antibodies to Clostridium difficileOrganisms in a Hen

Antibodies to certain pathogenic organisms have been shown to beeffective in treating diseases caused by those organisms. It has notbeen shown whether antibodies can be raised, against Clostridiumdifficile, which would be effective in treating infection by thisorganism. Accordingly, C. difficile was tested as immunogen forproduction of hen antibodies.

To determine the best course for raising high-titer egg antibodiesagainst whole C. difficile organisms, different immunizing strains anddifferent immunizing concentrations were examined. The example involved(a) preparation of the bacterial immunogen, (b) immunization, (c)purification of anti-bacterial chicken antibodies, and (d) detection ofanti-bacterial antibodies in the purified IgY preparations.

a) Preparation of Bacterial Immunogen

C. difficile strains 43594 (serogroup A) and 43596 (serogroup C) wereoriginally obtained from the ATCC. These two strains were selectedbecause they represent two of the most commonly-occurring serogroupsisolated from patients with antibiotic-associated pseudomembranouscolitis. [Delmee et al., J. Clin. Microbiol., 28(10):2210 (1990).]Additionally, both of these strains have been previously characterizedwith respect to their virulence in the Syrian hamster model for C.difficile infection. [Delmee et al., J. Med. Microbiol., 33:85 (1990).]

The bacterial strains were separately cultured on brain heart infusionagar for 48 hours at 37° C. in a Gas Pack 100 Jar (BBL, Cockeysville,Md.) equipped with a Gas Pack Plus anaerobic envelope (BBL). Forty-eighthour cultures were used because they produce better growth and theorganisms have been found to be more cross-reactive with respect totheir surface antigen presentation. The greater the degree ofcross-reactivity of our IgY preparations, the better the probability ofa broad range of activity against different strains/serogroups. [Toma etal., J. Clin. Microbiol., 26(3):426 (1988).]

The resulting organisms were removed from the agar surface using asterile dacron-tip swab, and were suspended in a solution containing0.4% formaldehyde in PBS, pH 7.2. This concentration of formaldehyde hasbeen reported as producing good results for the purpose of preparingwhole-organism immunogen suspensions for the generation of polyclonalanti-C. difficile antisera in rabbits. [Delmee et al., J. Clin.Microbiol., 21:323 (1985); Davies et al., Microbial Path., 9:141(1990).] In this manner, two separate bacterial suspensions wereprepared, one for each strain. The two suspensions were then incubatedat 4° C. for 1 hour. Following this period of formalin-treatment, thesuspensions were centrifuged at 4,200×g for 20 min., and the resultingpellets were washed twice in normal saline. The washed pellets, whichcontained formalin-treated whole organisms, were resuspended in freshnormal saline such that the visual turbidity of each suspensioncorresponded to a #7 McFarland standard. [M. A. C. Edelstein,“Processing Clinical Specimens for Anaerobic Bacteria: Isolation andIdentification Procedures,” in S. M. Finegold et al (eds.)., Bailey andScott's Diagnostic Microbiology, pp. 477-507, C. V. Mosby Co., (1990).The preparation of McFarland nephelometer standards and thecorresponding approximate number of organisms for each tube aredescribed in detail at pp. 172-173 of this volume.] Each of the two #7suspensions was then split into two separate volumes. One volume of eachsuspension was volumetrically adjusted, by the addition of saline, tocorrespond to the visual turbidity of a #1 McFarland standard. [Id.] The#1 suspensions contained approximately 3×10⁸ organisms/ml, and the #7suspensions contained approximately 2×10⁹ organisms/ml. [Id.] The fourresulting concentration-adjusted suspensions of formalin-treated C.difficile organisms were considered to be “bacterial immunogensuspensions.” These suspensions were used immediately after preparationfor the initial immunization. [See section (b).]

The formalin-treatment procedure did not result in 100% non-viablebacteria in the immunogen suspensions. In order to increase the level ofkilling, the formalin concentration and length of treatment were bothincreased for subsequent immunogen preparations, as described below inTable 3. (Although viability was decreased with the stronger formalintreatment, 100% inviability of the bacterial immunogen suspensions wasnot reached.) Also, in subsequent immunogen preparations, the formalinsolutions were prepared in normal saline instead of PBS. At day 49, theday of the fifth immunization, the excess volumes of the four previousbacterial immunogen suspensions were stored frozen at −70° C. for useduring all subsequent immunizations.

b) Immunization

For the initial immunization, 1.0 ml volumes of each of the fourbacterial immunogen suspensions described above were separatelyemulsified in 1.2 ml volumes of CFA (GIBCO). For each of the fouremulsified immunogen suspensions, two four-month old White Leghorn henspre-laying) were immunized. (It is not necessary to use pre-laying hens;actively-laying hens can also be utilized.) Each hen received a totalvolume of approximately 1.0 ml of a single emulsified immunogensuspension via four injections (two subcutaneous and two intramuscular)of approximately 250 μl per site. In this manner, a total of fourdifferent immunization combinations, using two hens per combination,were initiated for the purpose of evaluating both the effect ofimmunizing concentration on egg yolk antibody (IgY) production, andinterstrain cross-reactivity of IgY raised against heterologous strains.The four immunization groups are summarized in Table 3. TABLE 3Immunization Groups Approximate Group Designation Immunizing StrainImmunizing Dose CD 43594, #1 C. difficile strain 43594 1.5 × 10⁸organisms/hen CD 43594, #7 ″ 1.0 × 10⁹ organisms/hen CD 43596, #1 C.difficile strain 43596 1.5 × 10⁸ organisms/hen CD 43596, #7 ″ 1.0 × 10⁹organisms/hen

The time point for the first series of immunizations was designated as“day zero.” All subsequent immunizations were performed as describedabove except that the bacterial immunogen suspensions were emulsifiedusing IFA (GIBCO) instead of CFA, and for the later time pointimmunization, the stored frozen suspensions were used instead offreshly-prepared suspensions. The immunization schedule used is listedin Table 4. TABLE 4 Immunization Schedule Immunogen Day Of PreparationImmunization Formalin-Treatment Used 0 1%, 1 hr. freshly-prepared 14 1%,overnight ″ 21 1%, overnight ″ 35 1%, 48 hrs. ″ 49 1%, 72 hrs. ″ 70 ″stored frozen 85 ″ ″ 105 ″ ″

c) Purification of Anti-Bacterial Chicken Antibodies

Groups of four eggs were collected per immunization group between days80 and 84 post-initial immunization, and chicken immunoglobulin (IgY)was extracted according to a modification of the procedure of A. Polsonet al., Immunol. Comm., 9:495 (1980). A gentle stream of distilled waterfrom a squirt bottle was used to separate the yolks from the whites, andthe yolks were broken by dropping them through a funnel into a graduatedcylinder. The four individual yolks were pooled for each group. Thepooled, broken yolks were blended with 4 volumes of egg extractionbuffer to improve antibody yield (egg extraction buffer is 0.01 M sodiumphosphate, 0.1 M NaCl, pH 7.5, containing 0.005% thimerosal), and PEG8000 (Amresco) was added to a concentration of 3.5%. When all the PEGdissolved, the protein precipitates that formed were pelleted bycentrifugation at 13,000×g for 10 minutes. The supernatants weredecanted and filtered through cheesecloth to remove the lipid layer, andthe PEG was added to the supernatants to a final concentration of 12%(the supernatants were assumed to contain 3.5% PEG). After a secondcentrifugation, the supernatants were discarded and the pellets werecentrifuged a final time to extrude the remaining PEG. These crude IgYpellets were then dissolved in the original yolk volume of eggextraction buffer and stored at 4° C. As an additional control, apreimmune IgY solution was prepared as described above, using eggscollected from unimmunized hens.

d) Detection of Anti-Bacterial Antibodies in the Purified IgYPreparations

In order to evaluate the relative levels of specific anti-C. difficileactivity in the IgY preparations described above, a modified version ofthe whole-organism ELISA procedure of N. V. Padhye et al., J. Clin.Microbiol. 29:99-103 (1990) was used. Frozen organisms of both C.difficile strains described above were thawed and diluted to aconcentration of approximately 1×10⁷ organisms/ml using PBS, pH 7.2. Inthis way, two separate coating suspensions were prepared, one for eachimmunizing strain. Into the wells of 96-well microtiter plates (Falcon,Pro-Bind Assay Plates) were placed 100 μl volumes of the coatingsuspensions. In this manner, each plate well received a total ofapproximately 1×10⁶ organisms of one strain or the other. The plateswere then incubated at 4° C. overnight. The next morning, the coatingsuspensions were decanted, and all wells were washed three times usingPBS. In order to block non-specific binding sites, 100 μl of 0.5% BSA(Sigma) in PBS was then added to each well, and the plates wereincubated for 2 hours at room temperature. The blocking solution wasdecanted, and 100 μl volumes of the IgY preparations described abovewere initially diluted 1:500 with a solution of 0.1% BSA in PBS, andthen serially diluted in 1:5 steps. The following dilutions were placedin the wells: 1:500, 1:2,500, 1:62,5000, 1:312,500, and 1:1,562,500. Theplates were again incubated for 2 hours at room temperature. Followingthis incubation, the IgY-containing solutions were decanted, and thewells were washed three times using BBS-Tween (0.1 M boric acid, 0.025 Msodium borate, 1.0 M NaCl, 0.1% Tween-20), followed by two washes usingPBS-Tween (01% Tween-20), and finally, two washes using PBS only. Toeach well, 100 μl of a 1:750 dilution of rabbit anti-chicken IgG(whole-molecule)-alkaline phosphatase conjugate (Sigma) (diluted in 0.1%BSA in PBS) was added. The plates were again incubated for 2 hours atroom temperature. The conjugate solutions were decanted and the plateswere washed as described above, substituting 50 mM Na₂CO₃, pH 9.5 forthe PBS in the final wash. The plates were developed by the addition of100 μl of a solution containing 1 mg/ml para-nitrophenyl phosphate(Sigma) dissolved in 50 mM Na₂CO₃, 10 mM MgCl₂, pH 9.5 to each well, andincubating the plates at room temperature in the dark for 45 minutes.The absorbance of each well was measured at 410 nm using a Dynatech MR700 plate reader. In this manner, each of the four IgY preparationsdescribed above was tested for reactivity against both of the immunizingC. difficile strains; strain-specific, as well as cross-reactiveactivity was determined.

Table 5 shows the results of the whole-organism ELISA. All four IgYpreparations demonstrated significant levels of activity, to a dilutionof 1:62,500 or greater against both of the immunizing organism strains.Therefore, antibodies raised against one strain were highlycross-reactive with the other strain, and vice versa. The immunizingconcentration of organisms did not have a significant effect onorganism-specific IgY production, as both concentrations producedapproximately equivalent responses. Therefore, the lower immunizingconcentration of approximately 1.5×10⁸ organisms/hen is the preferredimmunizing concentration of the two tested. The preimmune IgYpreparation appeared to possess relatively low levels of C.difficile-reactive activity to a dilution of 1:500, probably due toprior exposure of the animals to environmental clostridia.

An initial whole-organism ELISA was performed using IgY preparationsmade from single CD 43594, #1 and CD 43596, #1 eggs collected around day50 (data not shown). Specific titers were found to be 5 to 10-fold lowerthan those reported in Table 5. These results demonstrate that it ispossible to begin immunizing hens prior to the time that they begin tolay eggs, and to obtain high titer specific IgY from the first eggs thatare laid. In other words, it is not necessary to wait for the hens tobegin laying before the immunization schedule is started. TABLE 5Results Of The Anti-C. difficile Whole-Organism ELISA Dilution Of IgY43594-Coated 43596-Coated IgY Preparation Prep Wells Wells CD 43594, #11:500 1.746 1.801 1:2,500 1.092 1.670 1:12,500 0.202 0.812 1:62,5000.136 0.179 1:312,500 0.012 0.080 1:1,562,500 0.002 0.020 CD 43594, #71:500 1.780 1.771 1:2,500 1.025 1.078 1:12,500 0.188 0.382 1:62,5000.052 0.132 1:312,500 0.022 0.043 1:1,562,500 0.005 0.024 CD 43596, #11:500 1.526 1.790 1:2,500 0.832 1.477 1:12,500 0.247 0.452 1:62,5000.050 0.242 1:312,500 0.010 0.067 1:1,562,500 0.000 0.036 CD 43596, #71:500 1.702 1.505 1:2,500 0.706 0.866 1:12,500 0.250 0.282 1:62,5000.039 0.078 1:312,500 0.002 0.017 1:1,562,500 0.000 0.010 Preimmune IgY1:500 0.142 0.309 1:2,500 0.032 0.077 1:12,500 0.006 0.024 1:62,5000.002 0.012 1:312,500 0.004 0.010 1:1,562,500 0.002 0.014

Example 2 Treatment of C. difficile Infection with Anti-C. difficileAntibody

In order to determine whether the immune IgY antibodies raised againstwhole C. difficile organisms were capable of inhibiting the infection ofhamsters by C. difficile, hamsters infected by these bacteria wereutilized. [Lyerly et al., Infect. Immun., 59:2215-2218 (1991).] Thisexample involved: (a) determination of the lethal dose of C. difficileorganisms; and (b) treatment of infected animals with immune antibody orcontrol antibody in nutritional solution.

a) Determination of the Lethal Dose of C. difficile Organisms

Determination of the lethal dose of C. difficile organisms was carriedout according to the model described by D. M. Lyerly et al., Infect.Immun., 59:2215-2218 (1991). C. difficile strain ATCC 43596 (serogroupC, ATCC) was plated on BHI agar and grown anaerobically (BBL Gas Pak 100system) at 37° C. for 42 hours. Organisms were removed from the agarsurface using a sterile dacron-tip swab and suspended in sterile 0.9%NaCl solution to a density of 108 organisms/ml.

In order to determine the lethal dose of C. difficile in the presence ofcontrol antibody and nutritional formula, non-immune eggs were obtainedfrom unimmunized hens and a 12% PEG preparation made as described inExample 1(c). This preparation was redissolved in one fourth theoriginal yolk volume of vanilla flavor Ensure®.

Starting on day one, groups of female Golden Syrian hamsters (HarlanSprague Dawley), 8-9 weeks old and weighing approximately 100 gm, wereorally administered 1 ml of the preimmune/Ensure® formula at time zero,2 hours, 6 hours, and 10 hours. At 1 hour, animals were orallyadministered 3.0 mg clindamycin HCl (Sigma) in 1 ml of water. This drugpredisposes hamsters to C. difficile infection by altering the normalintestinal flora. On day two, the animals were given 1 ml of thepreimmune IgY/Ensure® formula at time zero, 2 hours, 6 hours, and 10hours. At 1 hour on day two, different groups of animals were inoculatedorally with saline (control), or 10², 10⁴, 10⁶, or 10⁸ C. difficileorganisms in 1 ml of saline. From days 3-12, animals were given 1 ml ofthe preimmune IgY/Ensure® formula three times daily and observed for theonset of diarrhea and death. Each animal was housed in an individualcage and was offered food and water ad libitum.

Administration of 10⁶-10⁸ organisms resulted in death in 3-4 days whilethe lower doses of 10²-10⁴ organisms caused death in 5 days. Cecal swabstaken from dead animals indicated the presence of C. difficile. Giventhe effectiveness of the 10² dose, this number of organisms was chosenfor the following experiment to see if hyperimmune anti-C. difficileantibody could block infection.

b) Treatment of Infected Animals with Immune Antibody or ControlAntibody in Nutritional Formula

The experiment in (a) was repeated using three groups of seven hamsterseach. Group A received no clindamycin or C. difficile and was thesurvival control. Group B received clindamycin, 10² C. difficileorganisms and preimmune IgY on the same schedule as the animals in (a)above. Group C received clindamycin, 10² C. difficile organisms, andhyperimmune anti-C. difficile IgY on the same schedule as Group B. Theanti-C. difficile IgY was prepared as described in Example 1 except thatthe 12% PEG preparation was dissolved in one fourth the original yolkvolume of Ensure®.

All animals were observed for the onset of diarrhea or other diseasesymptoms and death. Each animal was housed in an individual cage and wasoffered food and water ad libitum. The results are shown in Table 6.TABLE 6 The Effect Of Oral Feeding Of Hyperimmune IgY Antibody on C.difficile Infection Time To Time To Animal Group Diarrhea^(a) Death^(a)A pre-immune IgY only no diarrhea no deaths B Clindamycin, C difficile,preimmune IgY 30 hrs. 49 hrs. C Clindamycin, C difficile, immune IgY 33hrs. 56 hrs.^(a)Mean of seven animals.

Hamsters in the control group A did not develop diarrhea and remainedhealthy during the experimental period. Hamsters in groups B and Cdeveloped diarrheal disease. Anti-C. difficile IgY did not protect theanimals from diarrhea or death, all animals succumbed in the same timeinterval as the animals treated with preimmune IgY. Thus, whileimmunization with whole organisms apparently can improve sub-lethalsymptoms with particular bacteria (see U.S. Pat. No. 5,080,895 to H.Tokoro), such an approach does not prove to be productive to protectagainst the lethal effects of C. difficile.

Example 3 Production of C. botulinum Type A Antitoxin in Hens

In order to determine whether antibodies could be raised against thetoxin produced by clostridial pathogens, which would be effective intreating clostridial diseases, antitoxin to C. botulinum type A toxinwas produced. This example involves: (a) toxin modification; (b)immunization; (c) antitoxin collection; (d) antigenicity assessment; and(e) assay of antitoxin titer.

a) Toxin Modification

C. botulinum type A toxoid was obtained from B. R. DasGupta. From this,the active type A neurotoxin (M. W. approximately 150 kD) was purifiedto greater than 99% purity, according to published methods. [B. R.DasGupta & V. Sathyamoorthy, Toxicon, 22:415 (1984).] The neurotoxin wasdetoxified with formaldehyde according to published methods. [B. R.Singh & B. R. DasGupta, Toxicon, 27:403 (1989).]

b) Immunization

C. botulinum toxoid for immunization was dissolved in PBS (1 mg/ml) andwas emulsified with an approximately equal volume of CFA (GIBCO) forinitial immunization or IFA for booster immunization. On day zero, twowhite leghorn hens, obtained from local breeders, were each injected atmultiple sites (intramuscular and subcutaneous) with 1 ml inactivatedtoxoid emulsified in 1 ml CFA. Subsequent booster immunizations weremade according to the following schedule for day of injection and toxoidamount: days 14 and 21-0.5 mg; day 171-0.75 mg; days 394, 401, 409-0.25mg. One hen received an additional booster of 0.150 mg on day 544.

c) Antitoxin Collection

Total yolk immunoglobulin (IgY) was extracted as described in Example1(c) and the IgY pellet was dissolved in the original yolk volume of PBSwith thimerosal.

d) Antigenicity Assessment

Eggs were collected from day 409 through day 423 to assess whether thetoxoid was sufficiently immunogenic to raise antibody. Eggs from the twohens were pooled and antibody was collected as described in the standardPEG protocol. [Example 1(c).] Antigenicity of the botulinal toxin wasassessed on Western blots. The 150 kD detoxified type A neurotoxin andunmodified, toxic, 300 kD botulinal type A complex (toxin used forintragastric route administration for animal gut neutralizationexperiments; see Example 6) were separated on a SDS-polyacrylamidereducing gel. The Western blot technique was performed according to themethod of Towbin. [H. Towbin et al., Proc. Natl. Acad. Sci. USA, 76:4350(1979).] Ten μg samples of C. botulinum complex and toxoid weredissolved in SDS reducing sample buffer (1% SDS, 0.5% 2-mercaptoethanol,50 mM Tris, pH 6.8, 10% glycerol, 0.025% w/v bromophenol blue, 10%β-mercaptoethanol), heated at 95° C. for 10 min and separated on a 1 mmthick 5% SDS-polyacrylamide gel. [K. Weber and M. Osborn, “Proteins andSodium Dodecyl Sulfate: Molecular Weight Determination on PolyacrylamideGels and Related Procedures,” in The Proteins, 3d Edition (H. Neurath &R. L. Hill, eds), pp. 179-223, (Academic Press, NY, 1975).] Part of thegel was cut off and the proteins were stained with Coomassie Blue. Theproteins in the remainder of the gel were transferred to nitrocelluloseusing the Milliblot-SDE electro-blotting system (Millipore) according tomanufacturer's directions. The nitrocellulose was temporarily stainedwith 10% Ponceau S [S. B. Carroll and A. Laughon, “Production andPurification of Polyclonal Antibodies to the Foreign Segment ofβ-galactosidase Fusion Proteins,” in DNA Cloning: A Practical Approach,Vol. III, (D. Glover, ed.), pp. 89-111, IRL Press, Oxford, (1987)] tovisualize the lanes, then distained by running a gentle stream ofdistilled water over the blot for several minutes. The nitrocellulosewas immersed in PBS containing 3% BSA overnight at 4° C. to block anyremaining protein binding sites.

The blot was cut into strips and each strip was incubated with theappropriate primary antibody. The avian anti-C. botulinum antibodies[described in (c)] and pre-immune chicken antibody (as control) werediluted 1:125 in PBS containing 1 mg/ml BSA for 2 hours at roomtemperature. The blots were washed with two changes each of largevolumes of PBS, BBS-Tween and PBS, successively (10 min/wash). Goatanti-chicken IgG alkaline phosphatase conjugated secondary antibody(Fisher Biotech) was diluted 1:500 in PBS containing 1 mg/ml BSA andincubated with the blot for 2 hours at room temperature. The blots werewashed with two changes each of large volumes of PBS and BBS-Tween,followed by one change of PBS and 0.1 M Tris-HCl, pH 9.5. Blots weredeveloped in freshly prepared alkaline phosphatase substrate buffer (100μg/ml nitroblue tetrazolium (Sigma), 50 μg/ml 5-bromo-4-chloro-3-indolylphosphate (Sigma), 5 mM MgCl₂ in 50 mM Na₂CO₃, pH 9.5).

The Western blots are shown in FIG. 1. The anti-C. botulinum IgY reactedto the toxoid to give a broad immunoreactive band at about 145-150 kD onthe reducing gel. This toxoid is refractive to disulfide cleavage byreducing agents due to formalin crosslinking. The immune IgY reactedwith the active toxin complex, a 97 kD C. botulinum type A heavy chainand a 53 kD light chain. The preimmune IgY was unreactive to the C.botulinum complex or toxoid in the Western blot.

e) Antitoxin Antibody Titer

The IgY antibody titer to C. botulinum type A toxoid of eggs harvestedbetween day 409 and 423 was evaluated by ELISA, prepared as follows.Ninety-six-well Falcon Pro-bind plates were coated overnight at 4° C.with 100 μl/well toxoid [B. R. Singh & B. R. Das Gupta, Toxicon 27:403(1989)] at 2.5 μg/ml in PBS, pH 7.5 containing 0.005% thimerosal. Thefollowing day the wells were blocked with PBS containing 1% BSA for 1hour at 37° C. The IgY from immune or preimmune eggs was diluted in PBScontaining 1% BSA and 0.05% Tween 20 and the plates were incubated for 1hour at 37° C. The plates were washed three times with PBS containing0.05% Tween 20 and three times with PBS alone. Alkalinephosphatase-conjugated goat-anti-chicken IgG (Fisher Biotech) wasdiluted 1:750 in PBS containing 1% BSA and 0.05% Tween 20, added to theplates, and incubated 1 hour at 37° C. The plates were washed as before,and p-nitrophenyl phosphate (Sigma) at 1 mg/ml in 0.05 M Na₂CO₃, pH 9.5,10 mM MgCl₂ was added.

The results are shown in FIG. 2. Chickens immunized with the toxoidgenerated high titers of antibody to the immunogen. Importantly, eggsfrom both immunized hens had significant anti-immunogen antibody titersas compared to preimmune control eggs. The anti-C. botulinum IgYpossessed significant activity, to a dilution of 1:93,750 or greater.

Example 4 Preparation of Avian Egg Yolk Immunoglobulin in an OrallyAdministrable Form

In order to administer avian IgY antibodies orally to experimental mice,an effective delivery formula for the IgY had to be determined. Theconcern was that if the crude IgY was dissolved in PBS, the saline inPBS would dehydrate the mice, which might prove harmful over theduration of the study. Therefore, alternative methods of oraladministration of IgY were tested. The example involved: (a) isolationof immune IgY; (b) solubilization of IgY in water or PBS, includingsubsequent dialysis of the IgY-PBS solution with water to eliminate orreduce the salts (salt and phosphate) in the buffer; and (c) comparisonof the quantity and activity of recovered IgY by absorbance at 280 nmand PAGE, and enzyme-linked immunoassay (ELISA).

a) Isolation of Immune IgY

In order to investigate the most effective delivery formula for IgY, weused IgY which was raised against Crotalus durissus terrificus venom.Three eggs were collected from hens immunized with the C. durissusterrificus venom and IgY was extracted from the yolks using the modifiedPolson procedure described by Thalley and Carroll [Bio/Technology,8:934-938 (1990)] as described in Example 1(c).

The egg yolks were separated from the whites, pooled, and blended withfour volumes of PBS. Powdered PEG 8000 was added to a concentration of3.5%. The mixture was centrifuged at 10,000 rpm for 10 minutes to pelletthe precipitated protein, and the supernatant was filtered throughcheesecloth to remove the lipid layer. Powdered PEG 8000 was added tothe supernatant to bring the final PEG concentration to 12% (assuming aPEG concentration of 3.5% in the supernatant). The 12% PEG/IgY mixturewas divided into two equal volumes and centrifuged to pellet the IgY.

b) Solubilization of the IgY in Water or PBS

One pellet was resuspended in ½ the original yolk volume of PBS, and theother pellet was resuspended in ½ the original yolk volume of water. Thepellets were then centrifuged to remove any particles or insolublematerial. The IgY in PBS solution dissolved readily but the fractionresuspended in water remained cloudy.

In order to satisfy anticipated sterility requirements for orallyadministered antibodies, the antibody solution needs to befilter-sterilized (as an alternative to heat sterilization which woulddestroy the antibodies). The preparation of IgY resuspended in water wastoo cloudy to pass through either a 0.2 or 0.45 μm membrane filter, so10 ml of the PBS resuspended fraction was dialyzed overnight at roomtemperature against 250 ml of water. The following morning the dialysischamber was emptied and refilled with 250 ml of fresh H₂O for a seconddialysis. Thereafter, the yields of soluble antibody were determined atOD₂₈₀ and are compared in Table 7. TABLE 7 Dependence of IgY Yield onSolvents Absorbance Of 1:10 Percent Fraction Dilution At 280 nm RecoveryPBS dissolved 1.149 100%  H₂O dissolved 0.706 61% PBS dissolved/H₂O0.885 77% dialyzed

Resuspending the pellets in PBS followed by dialysis against waterrecovered more antibody than directly resuspending the pellets in water(77% versus 61%). Equivalent volumes of the IgY preparation in PBS orwater were compared by PAGE, and these results were in accordance withthe absorbance values (data not shown).

c) Activity of IgY Prepared with Different Solvents

An ELISA was performed to compare the binding activity of the IgYextracted by each procedure described above. C. durissus terrificus(C.d.t.) venom at 2.5 μg/ml in PBS was used to coat each well of a96-well microtiter plate. The remaining protein binding sites wereblocked with PBS containing 5 mg/ml BSA. Primary antibody dilutions (inPBS containing 1 mg/ml BSA) were added in duplicate. After 2 hours ofincubation at room temperature, the unbound primary antibodies wereremoved by washing the wells with PBS, BBS-Tween, and PBS. The speciesspecific secondary antibody (goat anti-chicken immunoglobulinalkaline-phosphatase conjugate (Sigma) was diluted 1:750 in PBScontaining 1 mg/ml BSA and added to each well of the microtiter plate.After 2 hours of incubation at room temperature, the unbound secondaryantibody was removed by washing the plate as before, and freshlyprepared alkaline phosphatase substrate (Sigma) at 1 mg/ml in 50 mMNa₂CO₃, 10 mM MgCl₂, pH 9.5 was added to each well. The colordevelopment was measured on a Dynatech MR 700 microplate reader using a412 nm filter. The results are shown in Table 8.

The binding assay results parallel the recovery values in Table 7, withPBS-dissolved IgY showing slightly more activity than thePBS-dissolved/H₂O dialyzed antibody. The water-dissolved antibody hadconsiderably less binding activity than the other preparations.

Example 5 Survival of Antibody Activity after Passage Through theGastrointestinal Tract

In order to determine the feasibility of oral administration ofantibody, it was of interest to determine whether orally administeredIgY survived passage through the gastrointestinal tract. The exampleinvolved: (a) oral administration of specific immune antibody mixed witha nutritional formula; and (b) assay of antibody activity extracted fromfeces. TABLE 8 Antigen-Binding Activity Of IgY Prepared With DifferentSolvents Dilution Preimmune PBS Dissolved H₂O Dissolved PBS/H₂O 1:5000.005 1.748 1.577 1.742 1:2,500 0.004 0.644 0.349 0.606 1:12,500 0.0010.144 0.054 0.090 1:62,500 0.001 0.025 0.007 0.016 1:312,500 0.010 0.0000.000 0.002

a) Oral Administration of Antibody

The IgY preparations used in this example are the same PBS-dissolved/H₂Odialyzed antivenom materials obtained in Example 4 above, mixed with anequal volume of Enfamil®. Two mice were used in this experiment, eachreceiving a different diet as follows:

1) water and food as usual;

2) immune IgY preparation dialyzed against water and mixed 1:1 withEnfamil®. (The mice were given the corresponding mixture as their onlysource of food and water).

b) Antibody Activity after Ingestion

After both mice had ingested their respective fluids, each tube wasrefilled with approximately 10 ml of the appropriate fluid first thingin the morning. By mid-morning there was about 4 to 5 ml of liquid leftin each tube. At this point stool samples were collected from eachmouse, weighed, and dissolved in approximately 500 μl PBS per 100 mgstool sample. One hundred and sixty mg of control stools (no antibody)and 99 mg of experimental stools (specific antibody) in 1.5 ml microfugetubes were dissolved in 800 and 500 μl PBS, respectively. The sampleswere heated at 37° C. for 10 minutes and vortexed vigorously. Theexperimental stools were also broken up with a narrow spatula. Eachsample was centrifuged for 5 minutes in a microfuge and thesupernatants, presumably containing the antibody extracts, werecollected. The pellets were saved at 2-8° C. in case future extractswere needed. Because the supernatants were tinted, they were dilutedfive-fold in PBS containing 1 mg/ml BSA for the initial dilution in theenzyme immunoassay (ELISA). The primary extracts were then dilutedfive-fold serially from this initial dilution. The volume of primaryextract added to each well was 190 μl. The ELISA was performed exactlyas described in Example 4. TABLE 9 Specific Antibody Activity AfterPassage Through The Gastrointestinal Tract Control Fecal EXP. FecalDilution Preimmune IgY Extract Extract 1:5 <0 0.000 0.032 1:25 0.016 <00.016 1:125 <0 <0 0.009 1:625 <0 0.003 0.001 1:3125 <0 <0 0.000

There was some active antibody in the fecal extract from the mouse giventhe specific antibody in Enfamil® formula, but it was present at a verylow level. Since the samples were assayed at an initial 1:5 dilution,the binding observed could have been higher with less dilute samples.Consequently, the mice were allowed to continue ingesting either regularfood and water or the specific IgY in Enfamil® formula, as appropriate,so the assay could be repeated. Another ELISA plate was coated overnightwith 5 μg/ml of C.d.t. venom in PBS.

The following morning the ELISA plate was blocked with 5 mg/ml BSA, andthe fecal samples were extracted as before, except that instead ofheating the extracts at 37° C., the samples were kept on ice to limitproteolysis. The samples were assayed undiluted initially, and in 5×serial dilutions thereafter. Otherwise the assay was carried out asbefore. TABLE 10 Specific Antibody Survives Passage Through TheGastrointestinal Tract Control Dilution Preimmune IgY Extract Exp.Extract undiluted 0.003 <0 0.379 1:5 <0 <0 0.071 1:25 0.000 <0 0.0271:125 0.003 <0 0.017 1:625 0.000 <0 0.008 1:3125 0.002 <0 0.002

The experiment confirmed the previous results, with the antibodyactivity markedly higher. The control fecal extract showed noanti-C.d.t. activity, even undiluted, while the fecal extract from theanti-C.d.t. IgY/Enfamil®-fed mouse showed considerable anti-C.d.t.activity. This experiment (and the previous experiment) clearlydemonstrate that active IgY antibody survives passage through the mousedigestive tract, a finding with favorable implications for the successof IgY antibodies administered orally as a therapeutic or prophylactic.

Example 6 In Vivo Neutralization of Type C. botulinum Type A Neurotoxinby Avian Antitoxin Antibody

This example demonstrated the ability of PEG-purified antitoxin,collected as described in Example 3, to neutralize the lethal effect ofC. botulinum neurotoxin type A in mice. To determine the oral lethaldose (LD₁₀₀) of toxin A, groups of BALB/c mice were given differentdoses of toxin per unit body weight (average body weight of 24 grams).For oral administration, toxin A complex, which contains the neurotoxinassociated with other non-toxin proteins was used. This complex ismarkedly more toxic than purified neurotoxin when given by the oralroute. [I. Ohishi et al., Infect. Immun., 16:106 (1977).] C. botulinumtoxin type A complex, obtained from Eric Johnson (University OfWisconsin, Madison) was 250 μg/ml in 50 mM sodium citrate, pH 5.5,specific toxicity 3×10⁷ mouse LD₅₀/mg with parenteral administration.Approximately 40-50 ng/gm body weight was usually fatal within 48 hoursin mice maintained on conventional food and water. When mice were givena diet ad libitum of only Enfamil® the concentration needed to producelethality was approximately 2.5 times higher (125 ng/gm body weight).Botulinal toxin concentrations of approximately 200 ng/gm body weightwere fatal in mice fed Enfamil® containing preimmune IgY (resuspended inEnfamil® at the original yolk volume).

The oral LD₁₀₀ of C. botulinum toxin was also determined in mice thatreceived known amounts of a mixture of preimmune IgY-Ensure® deliveredorally through feeding needles. Using a 22 gauge feeding needle, micewere given 250 μl each of a preimmune IgY-Ensure® mixture (preimmune IgYdissolved in ¼ original yolk volume) 1 hour before and ½ hour and 5hours after administering botulinal toxin. Toxin concentrations givenorally ranged from approximately 12 to 312 ng/gm body weight (0.3 to 7.5μg per mouse). Botulinal toxin complex concentration of approximately 40ng/gm body weight (1 μg per mouse) was lethal in all mice in less than36 hours.

Two groups of BALB/c mice, 10 per group, were each given orally a singledose of 1 μg each of botulinal toxin complex in 100 μl of 50 mM sodiumcitrate pH 5.5. The mice received 250 μl treatments of a mixture ofeither preimmune or immune IgY in Ensure® (¼ original yolk volume) 1hour before and ½ hour, 4 hours, and 8 hours after botulinal toxinadministration. The mice received three treatments per day for two moredays. The mice were observed for 96 hours. The survival and mortalityare shown in Table 11. TABLE 11 Neutralization Of Botulinal Toxin A Invivo Toxin Dose Number Of Mice Number Of Mice ng/gm Antibody Type AliveDead 41.6 non-immune 0 10 41.6 anti-botulinal 10 0 toxin

All mice treated with the preimmune IgY-Ensure® mixture died within 46hours post-toxin administration. The average time of death in the micewas 32 hours post toxin administration. Treatments of preimmuneIgY-Ensure® mixture did not continue beyond 24 hours due to extensiveparalysis of the mouth in mice of this group. In contrast, all ten micetreated with the immune anti-botulinal toxin IgY-Ensure® mixturesurvived past 96 hours. Only 4 mice in this group exhibited symptoms ofbotulism toxicity (two mice about 2 days after and two mice 4 days aftertoxin administration). These mice eventually died 5 and 6 days later.Six of the mice in this immune group displayed no adverse effects to thetoxin and remained alive and healthy long term. Thus, the aviananti-botulinal toxin antibody demonstrated very good protection from thelethal effects of the toxin in the experimental mice.

Example 7

Production of an Avian Antitoxin Against Clostridium difficile Toxin A

Toxin A is a potent cytotoxin secreted by pathogenic strains of C.difficile, that plays a direct role in damaging gastrointestinaltissues. In more severe cases of C. difficile intoxication,pseudomembranous colitis can develop which may be fatal. This would beprevented by neutralizing the effects of this toxin in thegastrointestinal tract. As a first step, antibodies were producedagainst a portion of the toxin. The example involved: (a) conjugation ofa synthetic peptide of toxin A to bovine serum albumin; (b) immunizationof hens with the peptide-BSA conjugate; and (c) detection of antitoxinpeptide antibodies by ELISA.

a) Conjugation of a Synthetic Peptide of Toxin A to Bovine Serum Albumin

The synthetic peptide CQTIDGKKYYFN-NH₂ (SEQ ID NO:82) was preparedcommercially (Multiple Peptide Systems, San Diego, Calif.) and validatedto be 80% pure by high-pressure liquid chromatography. The eleven aminoacids following the cysteine residue represent a consensus sequence of arepeated amino acid sequence found in Toxin A. [Wren et al., Infect.Immun., 59:3151-3155 (1991).] The cysteine was added to facilitateconjugation to carrier protein.

In order to prepare the carrier for conjugation, BSA (Sigma) wasdissolved in 0.01 M NaPO₄, pH 7.0 to a final concentration of 20 mg/mland n-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Pierce) wasdissolved in N,N-dimethyl formamide to a concentration of 5 mg/ml. MBSsolution, 0.51 ml, was added to 3.25 ml of the BSA solution andincubated for 30 minutes at room temperature with stirring every 5minutes. The MBS-activated BSA was then purified by chromatography on aBio-Gel P-10 column (Bio-Rad; 40 ml bed volume) equilibrated with 50 mMNaPO₄, pH 7.0 buffer. Peak fractions were pooled (6.0 ml).

Lyophilized toxin A peptide (20 mg) was added to the activated BSAmixture, stirred until the peptide dissolved and incubated 3 hours atroom temperature. Within 20 minutes, the reaction mixture became cloudyand precipitates formed. After 3 hours, the reaction mixture wascentrifuged at 10,000×g for 10 min and the supernatant analyzed forprotein content. No significant protein could be detected at 280 nm. Theconjugate precipitate was washed three times with PBS and stored at 4°C. A second conjugation was performed with 15 mg of activated BSA and 5mg of peptide and the conjugates pooled and suspended at a peptideconcentration of 10 mg/ml in 10 mM NaPO₄, pH 7.2.

b) Immunization of Hens with Peptide Conjugate

Two hens were each initially immunized on day zero by injection into twosubcutaneous and two intramuscular sites with 1 mg of peptide conjugatethat was emulsified in CFA (GIBCO). The hens were boosted on day 14 andday 21 with 1 mg of peptide conjugate emulsified in IFA (GIBCO).

c) Detection of Antitoxin Peptide Antibodies by ELISA

IgY was purified from two eggs obtained before immunization (pre-immune)and two eggs obtained 31 and 32 days after the initial immunizationusing PEG fractionation as described in Example 1.

Wells of a 96-well microtiter plate (Falcon Pro-Bind Assay Plate) werecoated overnight at 4° C. with 100 μg/ml solution of the toxin Asynthetic peptide in PBS, pH 7.2 prepared by dissolving 1 mg of thepeptide in 1.0 ml of H₂O and dilution of PBS. The pre-immune and immuneIgY preparations were diluted in a five-fold series in a buffercontaining 1% PEG 8000 and 0.1% Tween-20 (v/v) in PBS, pH 7.2. The wellswere blocked for 2 hours at room temperature with 150 μl of a solutioncontaining 5% (v/v) Carnation® nonfat dry milk and 1% PEG 8000 in PBS,pH 7.2. After incubation for 2 hours at room temperature, the wells werewashed, secondary rabbit anti-chicken IgG-alkaline phosphatase (1:750)added, the wells washed again and the color development obtained asdescribed in Example 1. The results are shown in Table 12. TABLE 12Reactivity Of IgY With Toxin Peptide Absorbance At 410 NM Dilution OfPEG Prep Preimmune Immune Anti-Peptide 1:100 0.013 0.253 1:500 0.0040.039  1:2500 0.004 0.005

Clearly, the immune antibodies contain titers against this repeatedepitope of toxin A.

Example 8 Production of Avian Antitoxins Against Clostridium difficileNative Toxins A and B

To determine whether avian antibodies are effective for theneutralization of C. difficile toxins, hens were immunized using nativeC. difficile toxins A and B. The resulting egg yolk antibodies were thenextracted and assessed for their ability to neutralize toxins A and B invitro. The Example involved (a) preparation of the toxin immunogens, (b)immunization, (c) purification of the antitoxins, and (d) assay of toxinneutralization activity.

a) Preparation of the Toxin Immunogens

Both C. difficile native toxins A and B, and C. difficile toxoids,prepared by the treatment of the native toxins with formaldehyde, wereemployed as immunogens. C. difficile toxoids A and B were prepared by aprocedure which was modified from published methods (Ehrich et al.,Infect. Immun. 28:1041 (1980). Separate solutions (in PBS) of native C.difficile toxin A and toxin B (Tech Lab) were each adjusted to aconcentration of 0.20 mg/ml, and formaldehyde was added to a finalconcentration of 0.4%. The toxin/formaldehyde solutions were thenincubated at 37° C. for 40 hrs. Free formaldehyde was then removed fromthe resulting toxoid solutions by dialysis against PBS at 4° C. Inpreviously published reports, this dialysis step was not performed.Therefore, free formaldehyde must have been present in their toxoidpreparations. The toxoid solutions were concentrated, using a Centriprepconcentrator unit (Amicon), to a final toxoid concentration of 4.0mg/ml. The two resulting preparations were designated as toxoid A andtoxoid B.

C. difficile native toxins were prepared by concentrating stocksolutions of toxin A and toxin B (Tech Lab, Inc), using Centriprepconcentrator units (Amicon), to a final concentration of 4.0 mg/ml.

b) Immunization

The first two immunizations were performed using the toxoid A and toxoidB immunogens described above. A total of 3 different immunizationcombinations were employed. For the first immunization group, 0.2 ml oftoxoid A was emulsified in an equal volume of Titer Max adjuvant(CytRx). Titer Max was used in order to conserve the amount of immunogenused, and to simplify the immunization procedure. This immunizationgroup was designated “CTA.” For the second immunization group, 0.1 ml oftoxoid B was emulsified in an equal volume of Titer Max adjuvant. Thisgroup was designated “CTB.” For the third immunization group, 0.2 ml oftoxoid A was first mixed with 0.2 ml of toxoid B, and the resultingmixture was emulsified in 0.4 ml of Titer Max adjuvant. This group wasdesignated “CTAB.” In this way, three separate immunogen emulsions wereprepared, with each emulsion containing a final concentration of 2.0mg/ml of toxoid A (CTA) or toxoid B (CTB) or a mixture of 2.0 mg/mltoxoid A and 2.0 mg/ml toxoid B (CTAB).

On day 0, White Leghorn hens, obtained from a local breeder, wereimmunized as follows: Group CTA. Four hens were immunized, with each henreceiving 200 μg of toxoid A, via two intramuscular (I.M.) injections of50 μl of CTA emulsion in the breast area. Group CTB. One hen wasimmunized with 200 μg of toxoid B, via two I.M. injections of 50 μl ofCTB emulsion in the breast area. Group CTAB. Four hens were immunized,with each hen receiving a mixture containing 200 μg of toxoid A and 200μg of toxoid B, via two I.M. injections of 100 μl of CTAB emulsion inthe breast area. The second immunization was performed 5 weeks later, onday 35, exactly as described for the first immunization above.

In order to determine whether hens previously immunized with C.difficile toxoids could tolerate subsequent booster immunizations usingnative toxins, a single hen from group CTAB was immunized for a thirdtime, this time using a mixture of the native toxin A and native toxin Bdescribed in section (a) above (these toxins were notformaldehyde-treated, and were used in their active form). This was donein order to increase the amount (titer) and affinity of specificantitoxin antibody produced by the hen over that achieved by immunizingwith toxoids only. On day 62, 0.1 ml of a toxin mixture was preparedwhich contained 200 μg of native toxin A and 200% g of native toxin B.This toxin mixture was then emulsified in 0.1 ml of Titer Max adjuvant.A single CTAB hen was then immunized with the resulting immunogenemulsion, via two I.M. injections of 100 μl each, into the breast area.This hen was marked with a wing band, and observed for adverse effectsfor a period of approximately 1 week, after which time the hen appearedto be in good health.

Because the CTAB hen described above tolerated the booster immunizationwith native toxins A and B with no adverse effects, it was decided toboost the remaining hens with native toxin as well. On day 70, boosterimmunizations were performed as follows: Group CTA. A 0.2 ml volume ofthe 4 mg/ml native toxin A solution was emulsified in an equal volume ofTiter Max adjuvant. Each of the 4 hens was then immunized with 200 μg ofnative toxin A, as described for the toxoid A immunizations above. GroupCTB. A 50 μl volume of the 4 mg/ml native toxin B solution wasemulsified in an equal volume of Titer Max adjuvant. The hen was thenimmunized with 200 μg of native toxin B, as described for the toxoid Bimmunizations above. Group CTAB. A 0.15 ml volume of the 4 mg/ml nativetoxin A solution was first mixed with a 0.15 ml volume the 4 mg/mlnative toxin B solution. The resulting toxin mixture was then emulsifiedin 0.3 ml of Titer Max adjuvant. The 3 remaining hens (the hen with thewing band was not immunized this time) were then immunized with 200% gof native toxin A and 200% g of native toxin B as described for thetoxoid A+toxoid B immunizations (CTAB) above. On day 85, all hensreceived a second booster immunization using native toxins, done exactlyas described for the first boost with native toxins above.

All hens tolerated both booster immunizations with native toxins with noadverse effects. As previous literature references describe the use offormaldehyde-treated toxoids, this is apparently the first time that anyimmunizations have been performed using native C. difficile toxins.

c) Purification of Antitoxins

Eggs were collected from the hen in group CTB 10-12 days following thesecond immunization with toxoid (day 35 immunization described insection (b) above), and from the hens in groups CTA and CTAB 20-21 daysfollowing the second immunization with toxoid. To be used as apre-immune (negative) control, eggs were also collected from unimmunizedhens from the same flock. Egg yolk immunoglobulin (IgY) was extractedfrom the 4 groups of eggs as described in Example 1(c), and the finalIgY pellets were solubilized in the original yolk volume of PBS withoutthimerosal. Importantly, thimerosal was excluded because it would havebeen toxic to the CHO cells used in the toxin neutralization assaysdescribed in section (d) below.

d) Assay of Toxin Neutralization Activity

The toxin neutralization activity of the IgY solutions prepared insection (c) above was determined using an assay system that was modifiedfrom published methods. [Ehrich et al., Infect. Immun. 28:1041-1043(1992); and McGee et al. Microb. Path. 12:333-341 (1992).] As additionalcontrols, affinity-purified goat anti-C. difficile toxin A (Tech Lab)and affinity-purified goat anti-C. difficile toxin B (Tech Lab) werealso assayed for toxin neutralization activity.

The IgY solutions and goat antibodies were serially diluted using F 12medium (GIBCO) which was supplemented with 2% FCS (GIBCO) (this solutionwill be referred to as “medium” for the remainder of this Example). Theresulting antibody solutions were then mixed with a standardizedconcentration of either native C. difficile toxin A (Tech Lab), ornative C. difficile toxin B (Tech Lab), at the concentrations indicatedbelow. Following incubation at 37° C. for 60 min., 100 μl volumes of thetoxin+antibody mixtures were added to the wells of 96-well microtiterplates (Falcon Microtest III) which contained 2.5×10⁴ Chinese HamsterOvary (CHO) cells per well (the CHO cells were plated on the previousday to allow them to adhere to the plate wells). The final concentrationof toxin, or dilution of antibody indicated below refers to the finaltest concentration of each reagent present in the respective microtiterplate wells. Toxin reference wells were prepared which contained CHOcells and toxin A or toxin B at the same concentration used for thetoxin plus antibody mixtures (these wells contained no antibody).Separate control wells were also prepared which contained CHO cells andmedium only. The assay plates were then incubated for 18-24 hrs. in a37° C., humidified, 5% CO₂ incubator. On the following day, theremaining adherent (viable) cells in the plate wells were stained using0.2% crystal violet (Mallinckrodt) dissolved in 2% ethanol, for 10 min.Excess stain was then removed by rinsing with water, and the stainedcells were solubilized by adding 1001 μl of 1% SDS (dissolved in water)to each well. The absorbance of each well was then measured at 570 nm,and the percent cytotoxicity of each test sample or mixture wascalculated using the following formula:${\%\quad{CHO}\quad{Cell}{\quad\quad}{Cytotoxity}} = {\left\lbrack {1 - \frac{\left( {{Abs}.\quad{Sample}} \right)}{{Abs}.\quad{Control}}} \right\rbrack \times 100}$

Unlike previous reports which quantitate results visually by countingcell rounding by microscopy, this Example utilized spectrophotometricmethods to quantitate the C. difficile toxin bioassay. In order todetermine the toxin A neutralizing activity of the CTA, CTAB, andpre-immune IgY preparations, as well as the affinity-purified goatantitoxin A control, dilutions of these antibodies were reacted againsta 0.1 μg/ml concentration of native toxin A (this is the approx. 50%cytotoxic dose of toxin A in this assay system). The results are shownin FIG. 3.

Complete neutralization of toxin A occurred with the CTA IgY (antitoxinA, above) at dilutions of 1:80 and lower, while significantneutralization occurred out to the 1:320 dilution. The CTAB IgY(antitoxin A+toxin B, above) demonstrated complete neutralization at the1:320-1:160 and lower dilutions, and significant neutralization occurredout to the 1:1280 dilution. The commercially available affinity-purifiedgoat antitoxin A did not completely neutralize toxin A at any of thedilutions tested, but demonstrated significant neutralization out to adilution of 1:1,280. The preimmune IgY did not show any toxin Aneutralizing activity at any of the concentrations tested. These resultsdemonstrate that IgY purified from eggs laid by hens immunized withtoxin A alone, or simultaneously with toxin A and toxin B, is aneffective toxin A antitoxin.

The toxin B neutralizing activity of the CTAB and pre-immune IgYpreparations, and also the affinity-purified goat antitoxin B controlwas determined by reacting dilutions of these antibodies against aconcentration of native toxin B of 0.1 ng/ml (approximately the 50%cytotoxic dose of toxin B in the assay system). The results are shown inFIG. 4.

Complete neutralization of toxin B occurred with the CTAB IgY (antitoxinA+toxin B, above) at the 1:40 and lower dilutions, and significantneutralization occurred out to the 1:320 dilution. The affinity-purifiedgoat antitoxin B demonstrated complete neutralization at dilutions of1:640 and lower, and significant neutralization occurred out to adilution of 1:2,560. The preimmune IgY did not show any toxin Bneutralizing activity at any of the concentrations tested. These resultsdemonstrate that IgY purified from eggs laid by hens immunizedsimultaneously with toxin A and toxin B is an effective toxin Bantitoxin.

In a separate study, the toxin B neutralizing activity of CTB, CTAB, andpre-immune IgY preparations was determined by reacting dilutions ofthese antibodies against a native toxin B concentration of 0.1 μg/ml(approximately 100% cytotoxic dose of toxin B in this assay system). Theresults are shown in FIG. 5.

Significant neutralization of toxin B occurred with the CTB IgY(antitoxin B, above) at dilutions of 1:80 and lower, while the CTAB IgY(antitoxin A+toxin B, above) was found to have significant neutralizingactivity at dilutions of 1:40 and lower. The preimmune IgY did not showany toxin B neutralizing activity at any of the concentrations tested.These results demonstrate that IgY purified from eggs laid by hensimmunized with toxin B alone, or simultaneously with toxin A and toxinB, is an effective toxin B antitoxin.

Example 9 In Vivo Protection of Golden Syrian Hamsters from C. difficileDisease by Avian Antitoxins Against C. difficile Toxins A and B

The most extensively used animal model to study C. difficile disease isthe hamster. [Lyerly et al, Infect. Immun. 47:349-352 (1992).] Severalother animal models for antibiotic-induced diarrhea exist, but nonemimic the human form of the disease as closely as the hamster model. [R.Fekety, “Animal Models of Antibiotic-Induced Colitis,” in O. Zak and M.Sande (eds.), Experimental Models in Antimicrobial Chemotherapy, Vol. 2,pp. 61-72, (1986).] In this model, the animals are first predisposed tothe disease by the oral administration of an antibiotic, such asclindamycin, which alters the population of normally-occurringgastrointestinal flora (Fekety, at 61-72). Following the oral challengeof these animals with viable C. difficile organisms, the hamstersdevelop cecitis, and hemorrhage, ulceration, and inflammation areevident in the intestinal mucosa. [Lyerly et al., Infect. Immun.47:349-352 (1985).] The animals become lethargic, develop severediarrhea, and a high percentage of them die from the disease. [Lyerly etal., Infect. Immun. 47:349-352 (1985).] This model is therefore ideallysuited for the evaluation of therapeutic agents designed for thetreatment or prophylaxis of C. difficile disease.

The ability of the avian C. difficile antitoxins, described in Example 1above, to protect hamsters from C. difficile disease was evaluated usingthe Golden Syrian hamster model of C. difficile infection. The Exampleinvolved (a) preparation of the avian C. difficile antitoxins, (b) invivo protection of hamsters from C. difficile disease by treatment withavian antitoxins, and (c) long-term survival of treated hamsters.

a) Preparation of the Avian C. difficile Antitoxins

Eggs were collected from hens in groups CTA and CTAB described inExample 1(b) above. To be used as a pre-immune (negative) control, eggswere also purchased from a local supermarket. Egg yolk immunoglobulin(IgY) was extracted from the 3 groups of eggs as described in Example1(c), and the final IgY pellets were solubilized in one fourth theoriginal yolk volume of Ensure® nutritional formula.

b) In Vivo Protection of Hamsters Against C. difficile Disease byTreatment with Avian Antitoxins

The avian C. difficile antitoxins prepared in section (a) above wereevaluated for their ability to protect hamsters from C. difficiledisease using an animal model system which was modified from publishedprocedures. [Fekety, at 61-72; Borriello et al., J. Med. Microbiol.,24:53-64 (1987); Kim et al., Infect. Immun., 55:2984-2992 (1987);Borriello et al., J. Med. Microbiol., 25:191-196 (1988); Delmee andAvesani, J. Med. Microbiol., 33:85-90 (1990); and Lyerly et al., Infect.Immun. 59:2215-2218 (1991).] For the study, three separate experimentalgroups were used, with each group consisting of 7 female Golden Syrianhamsters (Charles River), approximately 10 weeks old and weighingapproximately 100 gms. each. The three groups were designated “CTA,”“CTAB” and “Pre-immune.” These designations corresponded to theantitoxin preparations with which the animals in each group weretreated. Each animal was housed in an individual cage, and was offeredfood and water ad libitum through the entire length of the study. On day1, each animal was orally administered 1.0 ml of one of the threeantitoxin preparations (prepared in section (a) above) at the followingtimepoints: 0 hrs., 4 hrs., and 8 hrs. On day 2, the day 1 treatment wasrepeated. On day 3, at the 0 hr. timepoint, each animal was againadministered antitoxin, as described above. At 1 hr., each animal wasorally administered 3.0 mg of clindamycin-HCl (Sigma) in 1 ml of water.This treatment predisposed the animals to infection with C. difficile.As a control for possible endogenous C. difficile colonization, anadditional animal from the same shipment (untreated) was alsoadministered 3.0 mg of clindamycin-HCl in the same manner. Thisclindamycin control animal was left untreated (and uninfected) for theremainder of the study. At the 4 hr. and 8 hr. timepoints, the animalswere administered antitoxin as described above. On day 4, at the 0 hr.timepoint, each animal was again administered antitoxin as describedabove. At 1 hr., each animal was orally challenged with 1 ml of C.difficile inoculum, which contained approx. 100 C. difficile strain43596 organisms in sterile saline. C. difficile strain 43596, which is aserogroup C strain, was chosen because it is representative of one ofthe most frequently-occurring serogroups isolated from patients withantibiotic-associated pseudomembranous colitis. [Delmee et al., J. Clin.Microbiol., 28:2210-2214 (1985).] In addition, this strain has beenpreviously demonstrated to be virulent in the hamster model ofinfection. [Delmee and Avesani, J. Med. Microbiol., 33:85-90 (1990).] Atthe 4 hr. and 8 hr. timepoints, the animals were administered antitoxinas described above. On days 5 through 13, the animals were administeredantitoxin 3× per day as described for day 1 above, and observed for theonset of diarrhea and death. On the morning of day 14, the final resultsof the study were tabulated. These results are shown in Table 13.

Representative animals from those that died in the Pre-Immune and CTAgroups were necropsied. Viable C. difficile organisms were cultured fromthe ceca of these animals, and the gross pathology of thegastrointestinal tracts of these animals was consistent with thatexpected for C. difficile disease (inflamed, distended, hemorrhagiccecum, filled with watery diarrhea-like material). In addition, theclindamycin control animal remained healthy throughout the entire studyperiod, therefore indicating that the hamsters used in the study had notpreviously been colonized with endogenous C. difficile organisms priorto the start of the study. Following the final antitoxin treatment onday 13, a single surviving animal from the CTA group, and also from theCTAB group, was sacrificed and necropsied. No pathology was noted ineither animal. TABLE 13 Treatment Results Treatment Group No.AnimalsSurviving No.Animals Dead Pre-Immune 1 6 CTA (Antitoxin A 5 2 only) CTAB(Antitoxin + Antitoxin 7 0 B)

Treatment of hamsters with orally-administered toxin A and toxin Bantitoxin (group CTAB) successfully protected 7 out of 7 (100%) of theanimals from C. difficile disease. Treatment of hamsters withorally-administered toxin A antitoxin (group CTA) protected 5 but of 7(71%) of these animals from C. difficile disease. Treatment usingpre-immune IgY was not protective against C. difficile disease, as only1 out of 7 (14%) of these animals survived. These results demonstratethat the avian toxin A antitoxin and the avian toxin A+toxin B antitoxineffectively protected the hamsters from C. difficile disease. Theseresults also suggest that although the neutralization of toxin A aloneconfers some degree of protection against C. difficile disease, in orderto achieve maximal protection, simultaneous antitoxin A and antitoxin Bactivity is necessary.

c) Long-Term Survival of Treated Hamsters

It has been previously reported in the literature that hamsters treatedwith orally-administered bovine antitoxin IgG concentrate are protectedfrom C. difficile disease as long as the treatment is continued, butwhen the treatment is stopped, the animals develop diarrhea andsubsequently die within 72 hrs. [Lyerly et al., Infect. Immun.,59(6):2215-2218 (1991).]

In order to determine whether treatment of C. difficile disease usingavian antitoxins promotes long-term survival following thediscontinuation of treatment, the 4 surviving animals in group CTA, andthe 6 surviving animals in group CTAB were observed for a period of 11days (264 hrs.) following the discontinuation of antitoxin treatmentdescribed in section (b) above. All hamsters remained healthy throughthe entire post-treatment period. This result demonstrates that not onlydoes treatment with avian antitoxin protect against the onset of C.difficile disease (i.e., it is effective as a prophylactic), it alsopromotes long-term survival beyond the treatment period, and thusprovides a lasting cure.

Example 10 In Vivo Treatment of Established C. difficile Infection inGolden Syrian Hamsters with Avian Antitoxins Against C. difficile ToxinsA and B

The ability of the avian C. difficile antitoxins, described in Example 8above, to treat an established C. difficile infection was evaluatedusing the Golden Syrian hamster model. The Example involved (a)preparation of the avian C. difficile antitoxins, (b) in vivo treatmentof hamsters with established C. difficile infection, and (c) histologicevaluation of cecal tissue.

a) Preparation of the Avian C. difficile Antitoxins

Eggs were collected from hens in group CTAB described in Example 8(b)above, which were immunized with C. difficile toxoids and native toxinsA and B. Eggs purchased from a local supermarket were used as apre-immune (negative) control. Egg yolk immunoglobulin (IgY) wasextracted from the 2 groups of eggs as described in Example 1(c), andthe final IgY pellets were solubilized in one-fourth the original yolkvolume of Ensure® nutritional formula.

b) In vivo Treatment of Hamsters with Established C. difficile Infection

The avian C. difficile antitoxins prepared in section (a) above wereevaluated for the ability to treat established C. difficile infection inhamsters using an animal model system which was modified from theprocedure which was described for the hamster protection study inExample 8(b) above.

For the study, four separate experimental groups were used, with eachgroup consisting of 7 female Golden Syrian hamsters (Charles River),approx. 10 weeks old, weighing approximately 100 gms. each. Each animalwas housed separately, and was offered food and water ad libitum throughthe entire length of the study.

On day 1 of the study, the animals in all four groups were eachpredisposed to C. difficile infection by the oral administration of 3.0mg of clindamycin-HCl (Sigma) in 1 ml of water.

On day 2, each animal in all four groups was orally challenged with 1 mlof C. difficile inoculum, which contained approximately 100 C. difficilestrain 43596 organisms in sterile saline. C. difficile strain 43596 waschosen because it is representative of one of the mostfrequently-occurring serogroups isolated from patients withantibiotic-associated pseudomembranous colitis. [Delmee et al., J. Clin.Microbiol., 28:2210-2214 (1990).] In addition, as this was the same C.difficile strain used in all of the previous Examples above, it wasagain used in order to provide experimental continuity.

On day 3 of the study (24 hrs. post-infection), treatment was startedfor two of the four groups of animals. Each animal of one group wasorally administered 1.0 ml of the CTAB IgY preparation (prepared insection (a) above) at the following timepoints: 0 hrs., 4 hrs., and 8hrs. The animals in this group were designated “CTAB-24.” The animals inthe second group were each orally administered 1.0 ml of the pre-immuneIgY preparation (also prepared in section (a) above) at the sametimepoints as for the CTAB group. These animals were designated“Pre-24.” Nothing was done to the remaining two groups of animals on day3.

On day 4, 48 hrs. post-infection, the treatment described for day 3above was repeated for the CTAB-24 and Pre-24 groups, and was initiatedfor the remaining two groups at the same timepoints. The final twogroups of animals were designated “CTAB-48” and “Pre-48” respectively.

On days 5 through 9, the animals in all four groups were administeredantitoxin or pre-immune IgY, 3× per day, as described for day 4 above.The four experimental groups are summarized in Table 14. TABLE 14Experimental Treatment Groups Group Designation Experimental TreatmentCTAB-24 Infected, treatment w/antitoxin IgY started @ 24 hrs. post-infection. Pre-24 Infected, treatment w/pre- immune IgY started @ 24hrs. post-infection. CTAB-48 Infected, treatment w/antitoxin IgY started@ 48 hrs. post- infection. Pre-48 Infected, treatment w/pre- immune IgYstarted @ 48 hrs. post-infection.

All animals were observed for the onset of diarrhea and death throughthe conclusion of the study on the morning of day 10. The results ofthis study are displayed in Table 15. TABLE 15 Experimental Outcome-Day10 Treatment No. Animals No. Animals Group Surviving Dead CTAB-24 6 1Pre-24 0 7 CTAB-48 4 3 Pre-48 2 5

Eighty-six percent of the animals which began receiving treatment withantitoxin IgY at 24 hrs. post-infection (CTAB-24 above) survived, while57% of the animals treated with antitoxin IgY starting 48 hrs.post-infection (CTAB-48 above) survived. In contrast, none of theanimals receiving pre-immune IgY starting 24 hrs. post-infection (Pre-24above) survived, and only 29% of the animals which began receivingtreatment with pre-immune IgY at 48 hrs. post-infection (Pre-48 above)survived through the conclusion of the study. These results demonstratethat avian antitoxins raised against C. difficile toxins A and B arecapable of successfully treating established C. difficile infections invivo.

c) Histologic Evaluation of Cecal Tissue

In order to further evaluate the ability of the IgY preparations testedin this study to treat established C. difficile infection, histologicevaluations were performed on cecal tissue specimens obtained fromrepresentative animals from the study described in section (b) above.

Immediately following death, cecal tissue specimens were removed fromanimals which died in the Pre-24 and Pre-48 groups. Following thecompletion of the study, a representative surviving animal wassacrificed and cecal tissue specimens were removed from the CTAB-24 andCTAB-48 groups. A single untreated animal from the same shipment asthose used in the study was also sacrificed and a cecal tissue specimenwas removed as a normal control. All tissue specimens were fixedovernight at 4° C. in 10% buffered formalin. The fixed tissues wereparaffin-embedded, sectioned, and mounted on glass microscope slides.The tissue sections were then stained using hematoxylin and eosin (H andE stain), and were examined by light microscopy.

Upon examination, the tissues obtained from the CTAB-24 and CTAB-48animals showed no pathology, and were indistinguishable from the normalcontrol. This observation provides further evidence for the ability ofavian antitoxins raised against C. difficile toxins A and B toeffectively treat established C. difficile infection, and to prevent thepathologic consequences which normally occur as a result of C. difficiledisease.

In contrast, characteristic substantial mucosal damage and destructionwas observed in the tissues of the animals from the Pre-24 and Pre-48groups which died from C. difficile disease. Normal tissue architecturewas obliterated in these two preparations, as most of the mucosal layerwas observed to have sloughed away, and there were numerous largehemorrhagic areas containing massive numbers of erythrocytes.

Example 11 Cloning and Expression of C. difficile Toxin A Fragments

The toxin A gene has been cloned and sequenced, and shown to encode aprotein of predicted MW of 308 kd. [Dove et al., Infect. Immun.,58:480-488 (1990).] Given the expense and difficulty of isolating nativetoxin A protein, it would be advantageous to use simple and inexpensiveprocaryotic expression systems to produce and purify high levels ofrecombinant toxin A protein for immunization purposes. Ideally, theisolated recombinant protein would be soluble in order to preservenative antigenicity, since solubilized inclusion body proteins often donot fold into native conformations. To allow ease of purification, therecombinant protein should be expressed to levels greater than 1mg/liter of E. coli culture.

To determine whether high levels of recombinant toxin A protein can beproduced in E. coli, fragments of the toxin A gene were cloned intovarious prokaryotic expression vectors, and assessed for the ability toexpress recombinant toxin A protein in E. coli. Three prokaryoticexpression systems were utilized. These systems were chosen because theydrive expression of either fusion (pMALc and pGEX2T) or native(pET23a-c) protein to high levels in E. coli, and allow affinitypurification of the expressed protein on a ligand containing column.Fusion proteins expressed from pGEX vectors bind glutathione agarosebeads, and are eluted with reduced glutathione. pMAL fusion proteinsbind amylose resin, and are eluted with maltose. A poly-histidine tag ispresent at either the N-terminal (pET16b) or C-terminal (pET23a-c) endof pET fusion proteins. This sequence specifically binds Ni₂ ⁺ chelatecolumns, and is eluted with imidazole salts. Extensive descriptions ofthese vectors are available [Williams et al. (1995) DNA Cloning 2:Expression Systems, Glover and Hames, eds. IRL Press, Oxford, pp.15-58], and will not be discussed in detail here. The Example involved(a) cloning of the toxin A gene, (b) expression of large fragments oftoxin A in various prokaryotic expression systems, (c) identification ofsmaller toxin A gene fragments that express efficiently in E. coli, (d)purification of recombinant toxin A protein by affinity chromatography,and (e) demonstration of functional activity of a recombinant fragmentof the toxin A gene.

a) Cloning of the Toxin A Gene

A restriction map of the toxin A gene is shown in FIG. 6. The encodedprotein contains a carboxy terminal ligand binding region, containingmultiple repeats of a carbohydrate binding domain. [von Eichel-Streiberand Sauerbom, Gene 96:107-113 (1990).] The toxin A gene was cloned inthree pieces, by using either the polymerase chain reaction (PCR) toamplify specific regions, (regions 1 and 2, FIG. 6) or by screening aconstructed genomic library for a specific toxin A gene fragment (region3, FIG. 6). The sequences of the utilized PCR primers are P1: 5′GGAAATTTAGCTGCAGCATCTGAC 3′ (SEQ ID NO.: 1); P2: 5′ TCTAGCAAATTCGCTTGTGTTGAA 3′ (SEQ ID NO:2); P3: 5′ CTCGCATATAGCATTAGACC 3′ (SEQ ID NO:3);and P4: 5′ CTATCTAGGCCTAAAGTAT 3′ (SEQ ID NO:4). These regions werecloned into prokaryotic expression vectors that express either fusion(pMALc and pGEX2T) or native (pET23a-c) protein to high levels in E.coli, and allow affinity purification of the expressed protein on aligand containing column.

Clostridium difficile VPI strain 10463 was obtained from the ATCC (ATCC#43255) and grown under anaerobic conditions in brain-heart infusionmedium (BBL). High molecular-weight C. difficile DNA was isolatedessentially as described by Wren and Tabaqchali (1987) J. Clin.Microbiol., 25:2402, except proteinase K and sodium dodecyl sulfate(SDS) was used to disrupt the bacteria, and cetyltrimethylammoniumbromide precipitation [as described in Ausubel et al., Current Protocolsin Molecular Biology (1989)] was used to remove carbohydrates from thecleared lysate. The integrity and yield of genomic DNA was assessed bycomparison with a serial dilution of uncut lambda DNA afterelectrophoresis on an agarose gel.

Fragments 1 and 2 were cloned by PCR, utilizing a proofreadingthermostable DNA polymerase (native pfu polymerase; Stratagene). Thehigh fidelity of this polymerase reduces the mutation problemsassociated with amplification by error prone polymerases (e.g., Taqpolymerase). PCR amplification was performed using the indicated PCRprimers (FIG. 6) in 50 μl reactions containing 10 mM Tris-HCl (8.3), 50mM KCl, 1.5 mM MgCl₂, 200 μM each dNTP, 0.2 μM each primer, and 50 ng C.difficile genomic DNA. Reactions were overlaid with 100 μl mineral oil,heated to 94° C. for 4 min, 0.5 μl native pfu polymerase (Stratagene)added, and the reaction cycled 30× at 94° C. for 1 min, 50° C. for 1min, 72° C. for 4 min, followed by 10 min at 72° C. Duplicate reactionswere pooled, chloroform extracted, and ethanol precipitated. Afterwashing in 70% ethanol, the pellets were resuspended in 50 μl TE buffer[10 mM Tris-HCL, 1 mM EDTA pH 8.0]. Aliquots of 101 μl each wererestriction digested with either EcoRI/HincII (fragment 1) or EcoRI/PstI(fragment 2), and the appropriate restriction fragments were gelpurified using the Prep-A-Gene kit (BioRad), and ligated to eitherEcoRI/SmaI-restricted pGEX2T (Pharmacia) vector (fragment 1), or theEcoRI/PstI pMA1c (New England Biolabs) vector (fragment 2). Both clonesare predicted to produce in-frame fusions with either theglutathione-S-transferase protein (PGEX vector) or the maltose bindingprotein (PMAL vector). Recombinant clones were isolated, and confirmedby restriction digestion, using standard recombinant molecular biologytechniques. [Sambrook et al., Molecular Cloning, A Laboratory Manual(1989), and designated pGA30-660 and pMA660-1100, respectively (see FIG.6 for description of the clone designations).]

Fragment 3 was cloned from a genomic library of size selected PstIdigested C. difficile genomic DNA, using standard molecular biologytechniques (Sambrook et al.). Given that the fragment 3 internal PstIsite is protected from cleavage in C. difficile genomic DNA [Price etal, Curr. Microbiol., 16:55-60 (1987)], a 4.7 kb fragment from PstIrestricted C. difficile genomic DNA was gel purified, and ligated toPstI restricted, phosphatase treated pUC9 DNA. The resulting genomiclibrary was screened with a oligonucleotide primer specific to fragment3, and multiple independent clones were isolated. The presence offragment 3 in several of these clones was confirmed by restrictiondigestion, and a clone of the indicated orientation (FIG. 6) wasrestricted with BamHI/HindIII, the released fragment purified by gelelectrophoresis, and ligated into similarly restricted pET23c expressionvector DNA (Novagen). Recombinant clones were isolated, and confirmed byrestriction digestion. This construct is predicted to create both apredicted in frame fusion with the pET protein leader sequence, as wellas a predicted C-terminal poly-histidine affinity tag, and is designatedpPA1100-2680 (see FIG. 6 for the clone designation).

b) Expression of Large Fragments of Toxin A in E. coli

Protein expression from the three expression constructs made in (a) wasinduced, and analyzed by Western blot analysis with an affinitypurified, goat polyclonal antiserum directed against the toxin A toxoid(Tech Lab). The procedures utilized for protein induction, SDS-PAGE, andWestern blot analysis are described in detail in Williams et al (1995),supra. In brief, 5 ml 2X YT (16 g tryptone, 10 g yeast extract, 5 g NaClper liter, pH 7.5+100 μg/ml ampicillin were added to cultures ofbacteria (BL21 for pMA1 and pGEX plasmids, and BL21(DE3)LysS for pETplasmids) containing the appropriate recombinant clone which wereinduced to express recombinant protein by addition of IPTG to 1 mM.Cultures were grown at 37° C., and induced when the cell density reached0.5 OD₆₀₀. Induced protein was allowed to accumulate for two hrs afterinduction. Protein samples were prepared by pelleting 1 ml aliquots ofbacteria by centrifugation (1 min in a microfuge), and resuspension ofthe pelleted bacteria in 150 μl of 2×SDS-PAGE sample buffer [Williams etal. (1995), supra]. The samples were heated to 95° C. for 5 min, thencooled and 5 or 10 μl aliquots loaded on 7.5% SDS-PAGE gels. BioRad highmolecular weight protein markers were also loaded, to allow estimationof the MW of identified fusion proteins. After electrophoresis, proteinwas detected either generally by staining gels with Coomassie blue, orspecifically, by blotting to nitrocellulose for Western blot detectionof specific immunoreactive protein. Western blots, (performed asdescribed in Example 3) which detect toxin A reactive protein in celllysates of induced protein from the three expression constructs areshown in FIG. 7. In this figure, lanes 1-3 contain cell lysates preparedfrom E. coli strains containing pPA1100-2860 in Bl21(DE3)lysE cells;lanes 4-6 contain cell lysates prepared from E. coli strains containingpPA1200-2860 in Bl21(DE3)lysS cells; lanes 7-9 contain cell lysatesprepared from E. coli strains containing pMA30-660; lanes 10-12 containcell lysates prepared from E. coli strains containing pMA660-1100. Thelanes were probed with an affinity purified goat antitoxin A polyclonalantibody (Tech Lab). Control lysates from uninduced cells (lanes 1, 7,and 10) contain very little immunoreactive material compared to theinduced samples in the remaining lanes. The highest molecular weightband observed for each clone is consistent with the predicted size ofthe full length fusion protein.

Each construct directs expression of high molecular weight (HMW) proteinthat is reactive with the toxin A antibody. The size of the largestimmunoreactive bands from each sample is consistent with predictions ofthe estimated MW of the intact fusion proteins. This demonstrates thatthe three fusions are in-frame, and that none of the clones containcloning artifacts that disrupt the integrity of the encoded fusionprotein. However, the Western blot demonstrates that fusion protein fromthe two larger constructs (pGA30-660 and pPA1100-2680) are highlydegraded. Also, expression levels of toxin A proteins from these twoconstructs are low, since induced protein bands are not visible byCoomassie staining (not shown). Several other expression constructs thatfuse large sub-regions of the toxin A gene to either pMALc or pET23a-cexpression vectors, were constructed and tested for protein induction.These constructs were made by mixing gel purified restriction fragments,derived from the expression constructs shown in FIG. 6, withappropriately cleaved expression vectors, ligating, and selectingrecombinant clones in which the toxin A restriction fragments hadligated together and into the expression vector as predicted forin-frame fusions. The expressed toxin A interval within these constructsare shown in FIG. 8, as well as the internal restriction sites utilizedto make these constructs.

As used herein, the term “interval” refers to any portion (i.e., anysegment of the toxin which is less than the whole toxin molecule) of aclostridial toxin. In a preferred embodiment, “interval” refers toportions of C. difficile toxins such as toxin A or toxin B. It is alsocontemplated that these intervals will correspond to epitopes ofimmunologic importance, such as antigens or immunogens against which aneutralizing antibody response is effected. It is not intended that thepresent invention be limited to the particular intervals or sequencesdescribed in these Examples. It is also contemplated that sub-portionsof intervals (e.g., an epitope contained within one interval or whichbridges multiple intervals) be used as compositions and in the methodsof the present invention.

In all cases, Western blot analysis of each of these constructs withgoat antitoxin A antibody (Tech Lab) detected HMW fusion protein of thepredicted size (not shown). This confirms that the reading frame of eachof these clones is not prematurely terminated, and is fused in thecorrect frame with the fusion partner. However, the Western blotanalysis revealed that in all cases, the induced protein is highlydegraded, and, as assessed by the absence of identifiable inducedprotein bands by Coomassie Blue staining, are expressed only at lowlevels. These results suggest that expression of high levels of intacttoxin A recombinant protein is not possible when large regions of thetoxin A gene are expressed in E. coli using these expression vectors.

c) High Level Expression of Small Toxin A Protein Fusions in E. coli

Experience indicates that expression difficulties are often encounteredwhen large (greater than 100 kd) fragments are expressed in E. coli. Anumber of expression constructs containing smaller fragments of thetoxin A gene were constructed, to determine if small regions of the genecan be expressed to high levels without extensive protein degradation. Asummary of these expression constructs are shown in FIG. 9. All wereconstructed by in-frame fusions of convenient toxin A restrictionfragments to either the pMALc or pET23a-c vectors. Protein preparationsfrom induced cultures of each of these constructs were analyzed by bothCoomassie Blue staining and Western analysis as in (b) above. In allcases, higher levels of intact, full length fusion proteins wereobserved than with the larger recombinants from section (b).

d) Purification of Recombinant Toxin A Protein

Large scale (500 ml) cultures of each recombinant from (c) were grown,induced, and soluble and insoluble protein fractions were isolated. Thesoluble protein extracts were affinity chromatographed to isolaterecombinant fusion protein, as described [Williams et al. (1994),supra]. In brief, extracts containing tagged pET fusions werechromatographed on a nickel chelate column, and eluted using imidazolesalts as described by the distributor (Novagen). Extracts containingsoluble pMAL fusion protein were prepared and chromatographed in columnbuffer (10 mM NaPO₄, 0.5M NaCl, 10 mM β-mercaptoethanol, pH 7.2) over anamylose resin column (New England Biolabs), and eluted with columnbuffer containing 10 mM maltose as described [Williams et al. (1995),supra]. When the expressed protein was found to be predominantlyinsoluble, insoluble protein extracts were prepared by the methoddescribed in Example 17, infra. The results are summarized in Table 16.FIG. 10 shows the sample purifications of recombinant toxin A protein.In this figure, lanes 1 and 2 contain MBP fusion protein purified byaffinity purification of soluble protein. TABLE 16 Purification OfRecombinant Toxin A Protein Yield Yield Affinity Intact PurifiedInsoluble Soluble % Intact Soluble Fusion Clone^((a)) Protein SolubilityProtein^((b)) Fusion Protein^((c)) Protein pMA30-270 Soluble   4 mg/500mls 10% NA PMA30-300 Soluble   4 mg/500 mls 5-10% NA pMA300-660Insoluble — NA 10 mg/500 ml pMA660-1100 Soluble  4.5 mg/500 mls 50% NApMA1100-1610 Soluble   18 mg/500 mls 10% NA pMA1610-1870 Both   22mg/500 mls 90% 20 mg/500 ml pMA1450-1870 Insoluble — NA 0.2 mg/500 ml pPA1100-1450 Soluble  0.1 mg/500 mls 90% NA pPA1100-1870 Soluble 0.02mg/500 mls 90% NA pMA1870-2680 Both   12 mg/500 mls 80% NA pPa1870-2680Insoluble — NA 10 mg/500 ml^((a))pP = pET23 vector, pM = pMALc vector, A = toxin A.^((b))Based on 1.5 OD₂₈₀ = 1 mg/ml (extinction coefficient of MBP).^((c))Estimated by Coomassie staining of SDS-PAGE gels.

Lanes 3 and 4 contain MBP fusion protein purified by solubilization ofinsoluble inclusion bodies. The purified fusion protein samples arepMA1870-2680 (lane 1), pMA660-1100 (lane 2), pMA300-600 (lane 3) andpMA1450-1870 (lane 4).

Poor yields of affinity purified protein were obtained whenpoly-histidine tagged pET vectors were used to drive expression(pPA1100-1450, pP1100-1870). However, significant protein yields wereobtained from pMAL expression constructs spanning the entire toxin Agene, and yields of full-length soluble fusion protein ranged from anestimated 200-400 μg/500 ml culture (pMA30-300) to greater than 20mg/500 ml culture (pMA1610-1870). Only one interval was expressed tohigh levels as strictly insoluble protein (pMA300-660). Thus, althoughhigh level expression was not observed when using large expressionconstructs from the toxin A gene, usable levels of recombinant proteinspanning the entire toxin A gene were obtainable by isolating inducedprotein from a series of smaller pMAL expression constructs that spanthe entire toxin A gene. This is the first demonstration of thefeasibility of expressing recombinant toxin A protein to high levels inE. coli.

e) Hemagglutination Assay Using the Toxin A Recombinant Proteins

The carboxy terminal end consisting of the repeating units contains thehemagglutination activity or binding domain of C. difficile toxin A. Todetermine whether the expressed toxin A recombinants retain functionalactivity, hemagglutination assays were performed. Two toxin Arecombinant proteins, one containing the binding domain as eithersoluble affinity purified protein (pMA1870-2680) or SDS solubilizedinclusion body protein (pPA1870-2680) and soluble protein from oneregion outside that domain (pMA1100-1610) were tested using a describedprocedure. [H. C. Krivan et. al., Infect. Immun., 53:573 (1986).]Citrated rabbit red blood cells (RRBC) (Cocalico) were washed severaltimes with Tris-buffer (0.1M Tris and 50 mM NaCl) by centrifugation at450×g for 10 minutes at 4° C. A 1% RRBC suspension was made from thepacked cells and resuspended in Tris-buffer. Dilutions of therecombinant proteins and native toxin A (Tech Labs) were made in theTris-buffer and added in duplicate to a round-bottomed 96-wellmicrotiter plate in a final volume of 100 μl. To each well, 50 μl of the1% RRBC suspension was added, mixed by gentle tapping, and incubated at4° C. for 3-4 hours. Significant hemagglutination occurred only in therecombinant proteins containing the binding domain (pMA1870-2680) andnative toxin A. The recombinant protein outside the binding domain(pMA1100-1610) displayed no hemagglutination activity. Using equivalentprotein concentrations, the hemagglutination titer for toxin A was1:256, while titers for the soluble and insoluble recombinant proteinsof the binding domain were 1:256 and about 1:5000. Clearly, therecombinant proteins tested retained functional activity and were ableto bind RRBC's.

Example 12 Functional Activity of IgY Reactive Against Toxin ARecombinants

The expression of recombinant toxin A protein as multiple fragments inE. coli has demonstrated the feasibility of generating toxin A antigenthrough use of recombinant methodologies (Example 11). The isolation ofthese recombinant proteins allows the immunoreactivity of eachindividual subregion of the toxin A protein to be determined (i.e., in aantibody pool directed against the native toxin A protein). Thisidentifies the regions (if any) for which little or no antibody responseis elicited when the whole protein is used as a immunogen. Antibodiesdirected against specific fragments of the toxin A protein can bepurified by affinity chromatography against recombinant toxin A protein,and tested for neutralization ability. This identifies any toxin Asubregions that are essential for producing neutralizing antibodies.Comparison with the levels of immune response directed against theseintervals when native toxin is used as an immunogen predicts whetherpotentially higher titers of neutralizing antibodies can be produced byusing recombinant protein directed against a individual region, ratherthan the entire protein. Finally, since it is unknown whether antibodiesreactive to the recombinant toxin A proteins produced in Example 11neutralize toxin A as effectively as antibodies raised against nativetoxin A (Examples 9 and 10), the protective ability of a pool ofantibodies affinity purified against recombinant toxin A fragments wasassessed for its ability to neutralize toxin A.

This Example involved (a) epitope mapping of the toxin A protein todetermine the titre of specific antibodies directed against individualsubregions of the toxin A protein when native toxin A protein is used asan immunogen, (b) affinity purification of IgY reactive againstrecombinant proteins spanning the toxin A gene, (c) toxin Aneutralization assays with affinity purified IgY reactive to recombinanttoxin A protein to identify subregions of the toxin A protein thatinduce the production of neutralizing antibodies, and determination ofwhether complete neutralization of toxin A can be elicited with amixture of antibodies reactive to recombinant toxin A protein.

a) Epitope Mapping of the Toxin A Gene

The affinity purification of recombinant toxin A protein specific todefined intervals of the toxin A protein allows epitope mapping ofantibody pools directed against native toxin A. This has not previouslybeen possible, since previous expression of toxin A recombinants hasbeen assessed only by Western blot analysis, without knowledge of theexpression levels of the protein [e.g., von Eichel-Streiber et al, J.Gen. Microbiol., 135:55-64 (1989)]. Thus, high or low reactivity ofrecombinant toxin A protein on Western blots may reflect proteinexpression level differences, not immunoreactivity differences. Giventhat the purified recombinant protein generated in Example 11 have beenquantitated, the issue of relative immunoreactivity of individualregions of the toxin A protein was precisely addressed.

For the purposes of this Example, the toxin A protein was subdividedinto 6 intervals (1-6), numbered from the amino (interval 1) to thecarboxyl (interval 6) termini.

The recombinant proteins corresponding to these intervals were fromexpression clones (see Example 11(d) for clone designations) pMA30-300(interval 1), pMA300-660 (interval 2), pMA660-1100 (interval 3),pPA1100-1450 (interval 4), pMA1450-1870 (interval 5) and pMA1870-2680(interval 6). These 6 clones were selected because they span the entireprotein from amino acids numbered 30 through 2680, and subdivide theprotein into 6 small intervals. Also, the carbohydrate binding repeatinterval is contained specifically in one interval (interval 6),allowing evaluation of the immune response specifically directed againstthis region. Western blots of 7.5% SDS-PAGE gels, loaded andelectrophoresed with defined quantities of each recombinant protein,were probed with either goat antitoxin A polyclonal antibody (Tech Lab)or chicken antitoxin A polyclonal antibody [pCTA IgY, Example 8(c)]. Theblots were prepared and developed with alkaline phosphatase aspreviously described [Williams et al. (1995), supra]. At least 90% ofall reactivity, in either goat or chicken antibody pools, was found tobe directed against the ligand binding domain (interval 6). Theremaining immunoreactivity was directed against all five remainingintervals, and was similar in both antibody pools, except that thechicken antibody showed a much lower reactivity against interval 2 thanthe goat antibody.

This clearly demonstrates that when native toxin A is used as animmunogen in goats or chickens, the bulk of the immune response isdirected against the ligand binding domain of the protein, with theremaining response distributed throughout the remaining ⅔ of theprotein.

b) Affinity Purification of IgY Reactive Against Recombinant Toxin AProtein

Affinity columns, containing recombinant toxin A protein from the 6defined intervals in (a) above, were made and used to (i) affinitypurify antibodies reactive to each individual interval from the CTA IgYpreparation [Example 8(c)], and (ii) deplete interval specificantibodies from the CTA IgY preparation. Affinity columns were made bycoupling 1 ml of PBS-washed Actigel resin (Sterogene) with regionspecific protein and 1/10 final volume of Ald-coupling solution (1Msodium cyanoborohydride). The total region specific protein added toeach reaction mixture was 2.7 mg F (interval 1), 3 mg (intervals 2 and3), 0.1 mg (interval 4), 0.2 mg (interval 5) and 4 mg (interval 6).Protein for intervals 1, 3, and 6 was affinity purified pMA1 fusionprotein in column buffer (see Example 11). Interval 4 was affinitypurified poly-histidine containing pET fusion in PBS; intervals 2 and 5were from inclusion body preparations of insoluble PMAL fusion protein,dialyzed extensively in PBS. Aliquots of the supernatants from thecoupling reactions, before and after coupling, were assessed byCoomassie staining of 7.5% SDS-PAGE gels. Based on protein bandintensities, in all cases greater than 50% coupling efficiencies wereestimated. The resins were poured into 5 ml BioRad columns, washedextensively with PBS, and stored at 4° C.

Aliquots of the CTA IgY polyclonal antibody preparation were depletedfor each individual region as described below. A 20 ml sample of the CTAIgY preparation [Example 8(c)] was dialyzed extensively against 3changes of PBS (1 liter for each dialysis), quantitated by absorbance atOD₂₈₀, and stored at 4° C. Six 1 ml aliquots of the dialyzed IgYpreparation were removed, and depleted individually for each of the sixintervals. Each 1 ml aliquot was passed over the appropriate affinitycolumn, and the eluate twice reapplied to the column. The eluate wascollected, and pooled with a 1 ml PBS wash. Bound antibody was elutedfrom the column by washing with 5 column volumes of 4 M Guanidine-HCl(in 10 mM Tris-HCl, pH 8.0). The column was reequilibrated in PBS, andthe depleted antibody stock reapplied as described above. The eluate wascollected, pooled with a 1 ml PBS wash, quantitated by absorbance atOD₂₈₀, and stored at 4° C. In this mariner, 6 aliquots of the CTA IgYpreparation were individually depleted for each of the 6 toxin Aintervals, by two rounds of affinity depletion. The specificity of eachdepleted stock was tested by Western blot analysis. Multiple 7.5%SDS-PAGE gels were loaded with protein samples corresponding to all 6toxin A subregions. After electrophoresis, the gels were blotted, andprotein transfer confirmed by Ponceau S staining [protocols described inWilliams et al. (1995), supra]. After blocking the blots 1 hr at 20° C.in PBS+0.1% Tween 20 (PBST) containing 5% milk (as a blocking buffer), 4ml of either a 1/500 dilution of the dialyzed CTA IgY preparation inblocking buffer, or an equivalent amount of the six depleted antibodystocks (using OD₂₈₀ to standardize antibody concentration) were addedand the blots incubated a further 1 hr at room temperature. The blotswere washed and developed with alkaline phosphatase (using a rabbitanti-chicken alkaline phosphate conjugate as a secondary antibody) aspreviously described [Williams et al. (1995), supra]. In all cases, onlythe target interval was depleted for antibody reactivity, and at least90% of the reactivity to the target intervals was specifically depleted.

Region specific antibody pools were isolated by affinity chromatographyas described below. Ten mls of the dialyzed CTA IgY preparation wereapplied sequentially to each affinity column, such that a single 10 mlaliquot was used to isolate region specific antibodies specific to eachof the six subregions. The columns were sequentially washed with 10volumes of PBS, 6 volumes of BBS-Tween, 10 volumes of TBS, and elutedwith 4 ml Actisep elution media (Sterogene). The eluate was dialyzedextensively against several changes of PBS, and the affinity purifiedantibody collected and stored at 4° C. The volumes of the eluateincreased to greater than 10 mls during dialysis in each case, due tothe high viscosity of the Actisep elution media. Aliquots of each samplewere 20× concentrated using Centricon 30 microconcentrators (Amicon) andstored at 4° C. The specificity of each region specific antibody poolwas tested, relative to the dialyzed CTA IgY preparation, by Westernblot analysis, exactly as described above, except that 4 ml samples ofblocking buffer containing 100 μl region specific antibody(unconcentrated) were used instead of the depleted CTA IgY preparations.Each affinity purified antibody preparation was specific to the definedinterval, except that samples purified against intervals 1-5 alsoreacted with interval 6. This may be due to non-specific binding to theinterval 6 protein, since this protein contains the repetitive ligandbinding domain which has been shown to bind antibodies nonspecifically.[Lyerly et al., Curr. Microbiol., 19:303-306 (1989).]

The reactivity of each affinity purified antibody preparation to thecorresponding proteins was approximately the same as the reactivity ofthe 1/500 diluted dialyzed CTA IgY preparation standard. Given that thespecific antibody stocks were diluted 1/40, this would indicate that theunconcentrated affinity purified antibody stocks contain 1/10- 1/20 theconcentration of specific antibodies relative to the starting CTA IgYpreparation.

c) Toxin A Neutralization Assay Using Antibodies Reactive TowardRecombinant Toxin A Protein

The CHO toxin neutralization assay [Example 8(d)] was used to assess theability of the depleted or enriched samples generated in (b) above toneutralize the cytotoxicity of toxin A. The general ability of affinitypurified antibodies to neutralize toxin A was assessed by mixingtogether aliquots of all 6 concentrated stocks of the 6 affinitypurified samples generated in (b) above, and testing the ability of thismixture to neutralize a toxin A concentration of 0.1 μg/ml. The results,shown in FIG. 11, demonstrate almost complete neutralization of toxin Ausing the affinity purified (AP) mix. Some epitopes within therecombinant proteins utilized for affinity purification were probablylost when the proteins were denatured before affinity purification [byGuanidine-HCl treatment in (b) above]. Thus, the neutralization abilityof antibodies directed against recombinant protein is probablyunderestimated using these affinity purified antibody pools. Thisexperiment demonstrates that antibodies reactive to recombinant toxin Acan neutralize cytotoxicity, suggesting that neutralizing antibodies maybe generated by using recombinant toxin A protein as immunogen.

In view of the observation that the recombinant expression clones of thetoxin A gene divide the protein into 6 subregions, the neutralizingability of antibodies directed against each individual region wasassessed. The neutralizing ability of antibodies directed against theligand binding domain of toxin A was determined first.

In the toxin neutralization experiment shown in FIG. 11, interval 6specific antibodies (interval 6 contains the ligand binding domain) weredepleted from the dialyzed PEG preparation, and the effect on toxinneutralization assayed. Interval 6 antibodies were depleted either byutilizing the interval 6 depleted CTA IgY preparation from (b) above(“−6 aff. depleted” in FIG. 11), or by addition of interval 6 protein tothe CTA IgY preparation (estimated to be a 10 fold molar excess overanti-interval 6 immunoglobulin present in this preparation) tocompetitively compete for interval 6 protein (“−6 prot depleted” in FIG.11). In both instances, removal of interval 6 specific antibodiesreduces the neutralization efficiency relative to the starting CTA IgYpreparation. This demonstrates that antibodies directed against interval6 contribute to toxin neutralization. Since interval 6 corresponds tothe ligand binding domain of the protein, these results demonstrate thatantibodies directed against this region in the PEG preparationcontribute to the neutralization of toxin A in this assay. However, itis significant that after removal of these antibodies, the PEGpreparation retains significant ability to neutralize toxin A (FIG. 11).This neutralization is probably due to the action of antibodies specificto other regions of the toxin A protein, since at least 90% of theligand binding region reactive antibodies were removed in the depletedsample prepared in (b) above. This conclusion was supported bycomparison of the toxin neutralization of the affinity purified (AP) mixcompared to affinity purified interval 6 antibody alone. Although someneutralization ability was observed with AP interval 6 antibodies alone,the neutralization was significantly less than that observed with themixture of all 6 AP antibody stocks (not shown).

Given that the mix of all six affinity purified samples almostcompletely neutralized the cytotoxicity of toxin A (FIG. 11), therelative importance of antibodies directed against toxin A intervals 1-5within the mixture was determined. This was assessed in two ways. First,samples containing affinity purified antibodies representing 5 of the 6intervals were prepared, such that each individual region was depletedfrom one sample. FIG. 12 demonstrates a sample neutralization curve,comparing the neutralization ability of affinity purified antibody mixeswithout interval 4 (−4) or 5 (−5) specific antibodies, relative to themix of all 6 affinity purified antibody stocks (positive control). Whilethe removal of interval 5 specific antibodies had no effect on toxinneutralization (or intervals 1-3, not shown), the loss of interval 4specific antibodies significantly reduced toxin neutralization (FIG.12).

Similar results were seen in a second experiment, in which affinitypurified antibodies, directed against a single region, were added tointerval 6 specific antibodies, and the effects on toxin neutralizationassessed. Only interval 4 specific antibodies significantly enhancedneutralization when added to interval 6 specific antibodies (FIG. 13).These results demonstrate that antibodies directed against interval 4(corresponding to clone pPA1100-1450 in FIG. 9) are important forneutralization of cytotoxicity in this assay. Epitope mapping has shownthat only low levels of antibodies reactive to this region are generatedwhen native toxin A is used as an immunogen [Example 12(a)]. It ishypothesized that immunization with recombinant protein specific to thisinterval will elicit higher titers of neutralizing antibodies. Insummary, this analysis has identified two critical regions of the toxinA protein against which neutralizing antibodies are produced, as assayedby the CHO neutralization assay.

Example 13 Production and Evaluation of Avian Antitoxin Against C.difficile Recombinant Toxin A Polypeptide

In Example 12, we demonstrated neutralization of toxin A mediatedcytotoxicity by affinity purified antibodies reactive to recombinanttoxin A protein. To determine whether antibodies raised against arecombinant polypeptide fragment of C. difficile toxin A may beeffective in treating clostridial diseases, antibodies to recombinanttoxin A protein representing the binding domain were generated. Twotoxin A binding domain recombinant polypeptides, expressing the bindingdomain in either the pMALc (pMA1870-2680) or pET 23(pPA1870-2680)vector, were used as immunogens. The pMAL protein was affinity purifiedas a soluble product [Example 12(d)] and the pET protein was isolated asinsoluble inclusion bodies [Example 12(d)] and solubilized to animmunologically active protein using a proprietary method described in apending patent application (U.S. patent application Ser. No.08/129,027). This Example involves (a) immunization, (b) antitoxincollection, (c) determination of antitoxin antibody titer, (d)anti-recombinant toxin A neutralization of toxin A hemagglutinationactivity in vitro, and (e) assay of in vitro toxin A neutralizingactivity.

a) Immunization

The soluble and the inclusion body preparations each were usedseparately to immunize hens. Both purified toxin A polypeptides werediluted in PBS and emulsified with approximately equal volumes of CFAfor the initial immunization or IFA for subsequent boosterimmunizations. On day zero, for each of the recombinant preparations,two egg laying white Leghorn hens (obtained from local breeder) wereeach injected at multiple sites (intramuscular and subcutaneous) with 1ml of recombinant adjuvant mixture containing approximately 0.5 to 1.5mgs of recombinant toxin A. Booster immunizations of 1.0 mg were givenon days 14 and day 28.

b) Antitoxin Collection

Total yolk immune IgY was extracted as described in the standard PEGprotocol (as in Example 1) and the final IgY pellet was dissolved insterile PBS at the original yolk volume. This material is designated“immune recombinant IgY” or “immune IgY.”

c) Antitoxin Antibody Titer

To determine if the recombinant toxin A protein was sufficientlyimmunogenic to raise antibodies in hens, the antibody titer of arecombinant toxin A polypeptide was determined by ELISA. Eggs from bothhens were collected on day 32, the yolks pooled and the antibody wasisolated using PEG as described. The immune recombinant IgY antibodytiter was determined for the soluble recombinant protein containing themaltose binding protein fusion generated in p-Mal (pMA1870-2680).Ninety-six well Falcon Pro-bind plates were coated overnight at 4° C.with 100 μL/well of toxin A recombinant at 2.5 μg/μl in PBS containing0.05% thimerosal. Another plate was also coated with maltose bindingprotein (MBP) at the same concentration, to permit comparison ofantibody reactivity to the fusion partner. The next day, the wells wereblocked with PBS containing 1% bovine serum albumin (BSA) for 1 hour at37° C. IgY isolated from immune or preimmune eggs was diluted inantibody diluent (PBS containing 1% BSA and 0.05% Tween-20), and addedto the blocked wells and incubated for 1 hour at 37° C. The plates werewashed three times with PBS with 0.05% Tween-20, then three times withPBS. Alkaline phosphatase conjugated rabbit anti-chicken IgG (Sigma)diluted 1:1000 in antibody diluent was added to the plate, and incubatedfor 1 hour at 37° C. The plates were washed as before and substrate wasadded, [p-nitrophenyl phosphate (Sigma)] at 1 mg/ml in 0.05M Na₂CO₃, pH9.5 and 10 mM MgCl₂. The plates were evaluated quantitatively on aDynatech MR 300 Micro EPA plate reader at 410 nm about 10 minutes afterthe addition of substrate.

Based on these ELISA results, high antibody titers were raised inchickens immunized with the toxin A recombinant polypeptide. Therecombinant appeared to be highly immunogenic, as it was able togenerate high antibody titers relatively quickly with few immunizations.Immune IgY titer directed specifically to the toxin A portion of therecombinant was higher than the immune IgY titer to its fusion partner,the maltose binding protein, and significantly higher than the preimmuneIgY. ELISA titers (reciprocal of the highest dilution of IgY generatinga signal) in the preimmune IgY to the MBP or the recombinant was <1:30while the immune IgY titers to MBP and the toxin A recombinant were1:18750 and >1:93750 respectively. Importantly, the anti-recombinantantibody titers generated in the hens against the recombinantpolypeptide is much higher, compared to antibodies to that region raisedusing native toxin A. The recombinant antibody titer to region 1870-2680in the CTA antibody preparation is at least five-fold lower compared tothe recombinant generated antibodies (1:18750 versus >1:93750). Thus, itappears a better immune response can be generated against a specificrecombinant using that recombinant as the immunogen compared to thenative toxin A.

This observation is significant, as it shows that because recombinantportions stimulate the production of antibodies, it is not necessary touse native toxin molecules to produce antitoxin preparations. Thus, theproblems associated with the toxicity of the native toxin are avoidedand large-scale antitoxin production is facilitated.

d) Anti-Recombinant Toxin A Neutralization of Toxin A HemagglutinationActivity In Vitro

Toxin A has hemagglutinating activity besides cytotoxic and enterotoxinproperties. Specifically, toxin A agglutinates rabbit erythrocytes bybinding to a trisaccharide (gal 1-3B1-4GlcNAc) on the cell surface. [H.Krivan et al., Infect. Immun., 53:573-581 (1986).] We examined whetherthe anti-recombinant toxin A (immune IgY, antibodies raised against theinsoluble product expressed in pET) can neutralize the hemagglutinationactivity of toxin A in vitro. The hemagglutination assay procedure usedwas described by H. C. Krivan et al. Polyethylene glycol-fractionatedimmune or preimmune IgY were pre-absorbed with citrated rabbiterythrocytes prior to performing the hemagglutination assay because wehave found that IgY alone can agglutinate red blood cells. Citratedrabbit red blood cells (RRBC's) (Cocalico) were washed twice bycentrifugation at 450×g with isotonic buffer (0.1 M Tris-HCl, 0.05 MNaCl, pH 7.2). RRBC-reactive antibodies in the IgY were removed bypreparing a 10% RRBC suspension (made by adding packed cells to immuneor preimmune IgY) and incubating the mixture for 1 hour at 37° C. TheRRBCs were then removed by centrifugation. Neutralization of thehemagglutination activity of toxin A by antibody was tested inround-bottomed 96-well microtiter plates. Twenty-five μl of toxin A (36μg/ml) (Tech Lab) in isotonic buffer was mixed with an equal volume ofdifferent dilutions of immune or preimmune IgY in isotonic buffer, andincubated for 15 minutes at room temperature. Then, 50 μl of a 1% RRBCsuspension in isotonic buffer was added and the mixture was incubatedfor 3 hours at 4° C. Positive control wells containing the finalconcentration of 9 μg/ml of toxin A after dilution without IgY were alsoincluded. Hemagglutination activity was assessed visually, with adiffuse matrix of RRBC's coating the bottom of the well representing apositive hemagglutination reaction and a tight button of RRBC's at thebottom of the well representing a negative reaction. Theanti-recombinant immune IgY neutralized toxin A hemagglutinationactivity, giving a neutralization titer of 1:8. However, preimmune IgYwas unable to neutralize the hemagglutination ability of toxin A.

e) Assay of In Vitro Toxin A Neutralizing Activity

The ability of the anti-recombinant toxin A IgY (immune IgY antibodiesraised against pMA1870-2680, the soluble recombinant binding domainprotein expressed in PMAL, designated as Anti-tox. A-2 in FIG. 14, andreferred to as recombinant region 6) and pre-immune IgY, prepared asdescribed in Example 8(c) above, to neutralize the cytotoxic activity oftoxin A was assessed in vitro using the CHO cell cytotoxicity assay, andtoxin A (Tech Lab) at a concentration of 0.1 μg/ml, as described inExample 8(d) above. As additional controls, the anti-native toxin A IgY(CTA) and pre-immune IgY preparations described in Example 8(c) abovewere also tested. The results are shown in FIG. 14.

The anti-recombinant toxin A IgY demonstrated only partialneutralization of the cytotoxic activity of toxin A, while thepre-immune IgY did not demonstrate any significant neutralizingactivity.

Example 14 In Vivo Neutralization of C. difficile Toxin A

The ability of avian antibodies (IgY) raised against recombinant toxin Abinding domain to neutralize the enterotoxin activity of C. difficiletoxin A was evaluated in vivo using Golden Syrian hamsters. The Exampleinvolved: (a) preparation of the avian anti-recombinant toxin A IgY fororal administration; (b) in vivo protection of hamsters from C.difficile toxin A enterotoxicity by treatment of toxin A with aviananti-recombinant toxin A IgY; and (c) histologic evaluation of hamsterceca.

a) Preparation of the Avian Anti-Recombinant Toxin A IgY for OralAdministration

Eggs were collected from hens which had been immunized with therecombinant C. difficile toxin A fragment pMA1870-2680 (described inExample 13, above). A second group of eggs purchased at a localsupermarket was used as a pre-immune (negative) control. Egg yolkimmunoglobulin (IgY) was extracted by PEG from the two groups of eggs asdescribed in Example 8(c), and the final IgY pellets were solubilized inone-fourth the original yolk volume using 0.1M carbonate buffer (mixtureof NaHCO₃ and Na₂CO₃), pH 9.5. The basic carbonate buffer was used inorder to protect the toxin A from the acidic pH of the stomachenvironment.

b) In Vivo Protection of Hamsters Against C. difficile Toxin AEnterotoxicity by Treatment of Toxin A with Avian Anti-Recombinant ToxinA IgY

In order to assess the ability of the avian anti-recombinant toxin AIgY, prepared in section (a) above to neutralize the in vivo enterotoxinactivity of toxin A, an in vivo toxin neutralization model was developedusing Golden Syrian hamsters. This model was based on published valuesfor the minimum amount of toxin A required to elicit diarrhea (0.08 mgtoxin A/Kg body wt.) and death (0.16 mg toxin A/Kg body wt.) in hamsterswhen administered orally (Lyerly et al. Infect. Immun., 47:349-352(1985).

For the study, four separate experimental groups were used, with eachgroup consisting of 7 female Golden Syrian hamsters (Charles River),approx. three and one-half weeks old, weighing approx. 50 gms each. Theanimals were housed as groups of 3 and 4, and were offered food andwater ad libitum through the entire length of the study.

For each animal, a mixture containing either 100 μg of toxin A (0.2mg/Kg) or 30 μg of toxin A (0.6 mg/Kg) (C. difficile toxin A wasobtained from Tech Lab and 1 ml of either the anti-recombinant toxin AIgY or pre-immune IgY (from section (a) above) was prepared. Thesemixtures were incubated at 37° C. for 60 min. and were then administeredto the animals by the oral route. The animals were then observed for theonset of diarrhea and death for a period of 24 hrs. following theadministration of the toxin A+IgY mixtures, at the end of which time,the following results were tabulated and shown in Table 17: TABLE 17Study Outcome At 24 Hours Study Outcome at 24 Hours Experimental GroupHealthy¹ Diarrhea² Dead³ 10 μg Toxin A + Antitoxin 7 0 0 AgainstInterval 6 30 μg Toxin A + Antitoxin 7 0 0 Against Interval 6 10 μgToxin A + Pre-Immune 0 5 2 Serum 30 μg Toxin A + Pre-Immune 0 5 2¹Animals remained healthy through the entire 24 hour study period.²Animals developed diarrhea, but did not die.³Animals developed diarrhea, and subsequently died.

Pretreatment of toxin A at both doses tested, using the anti-recombinanttoxin A IgY, prevented all overt symptoms of disease in hamsters.Therefore, pretreatment of C. difficile toxin A, using theanti-recombinant toxin A IgY, neutralized the in vivo enterotoxinactivity of the toxin A. In contrast, all animals from the two groupswhich received toxin A which had been pretreated using pre-immune IgYdeveloped disease symptoms which ranged from diarrhea to death. Thediarrhea which developed in the animals which did not die in each of thetwo pre-immune groups, spontaneously resolved by the end of the 24 hr.study period.

c) Histologic Evaluation of Hamster Ceca

In order to further assess the ability of anti-recombinant toxin A IgYto protect hamsters from the enterotoxin activity of toxin A, histologicevaluations were performed on the ceca of hamsters from the studydescribed in section (b) above.

Three groups of animals were sacrificed in order to prepare histologicalspecimens. The first group consisted of a single representative animaltaken from each of the 4 groups of surviving hamsters at the conclusionof the study described in section (b) above. These animals representedthe 24 hr. timepoint of the study.

The second group consisted of two animals which were not part of thestudy described above, but were separately treated with the same toxinA+pre-immune IgY mixtures as described for the animals in section (b)above. Both of these hamsters developed diarrhea, and were sacrificed 8hrs. after the time of administration of the toxin A+pre-immune IgYmixtures. At the time of sacrifice, both animals were presentingsymptoms of diarrhea. These animals represented the acute phase of thestudy.

The final group consisted of a single untreated hamster from the sameshipment of animals as those used for the two previous groups. Thisanimal served as the normal control.

Samples of cecal tissue were removed from the 7 animals described above,and were fixed overnight at 4° C. using 10% buffered formalin. The fixedtissues were paraffin-embedded, sectioned, and mounted on glassmicroscope slides. The tissue sections were then stained usinghematoxylin and eosin (H and E stain), and were examined by lightmicroscopy.

The tissues obtained from the two 24 hr. animals which received mixturescontaining either 10 μg or 30 μg of toxin A and anti-recombinant toxin AIgY were indistinguishable from the normal control, both in terms ofgross pathology, as well as at the microscopic level. These observationsprovide further evidence for the ability of anti-recombinant toxin A IgYto effectively neutralize the in vivo enterotoxin activity of C.difficile toxin A, and thus its ability to prevent acute or lastingtoxin A-induced pathology.

In contrast, the tissues from the two 24 hr. animals which received thetoxin A+pre-immune IgY mixtures demonstrated significant pathology. Inboth of these groups, the mucosal layer was observed to be lessorganized than in the normal control tissue. The cytoplasm of theepithelial cells had a vacuolated appearance, and gaps were presentbetween the epithelium and the underlying cell layers. The laminapropria was largely absent. Intestinal villi and crypts weresignificantly diminished, and appeared to have been overgrown by aplanar layer of epithelial cells and fibroblasts. Therefore, althoughthese animals overtly appeared to recover from the acute symptoms oftoxin A intoxication, lasting pathologic alterations to the cecal mucosahad occurred.

The tissues obtained from the two acute animals which received mixturesof toxin A and pre-immune IgY demonstrated the most significantpathology. At the gross pathological level, both animals were observedto have severely distended ceca which were filled with watery,diarrhea-like material. At the microscopic level, the animal that wasgiven the mixture containing 10 μg of toxin A and pre-immune IgY wasfound to have a mucosal layer which had a ragged, damaged appearance,and a disorganized, compacted quality. The crypts were largely absent,and numerous breaks in the epithelium had occurred. There was also aninflux of erythrocytes into spaces between the epithelial layer and theunderlying tissue. The animal which had received the mixture containing30 μg of toxin A and pre-immune IgY demonstrated the most severepathology. The cecal tissue of this animal had an appearance verysimilar to that observed in animals which had died from C. difficiledisease. Widespread destruction of the mucosa was noted, and theepithelial layer had sloughed. Hemorrhagic areas containing largenumbers of erythrocytes were very prevalent. All semblance of normaltissue architecture was absent from this specimen. In terms of thepresentation of pathologic events, this in vivo hamster model of toxinA-intoxication correlates very closely with the pathologic consequencesof C. difficile disease in hamsters. The results presented in thisExample demonstrate that while anti-recombinant toxin A (Interval 6) IgYis capable of only partially neutralizing the cytotoxic activity of C.difficile toxin A, the same antibody effectively neutralizes 100% of thein vivo enterotoxin activity of the toxin. While it is not intended thatthis invention be limited to this mechanism, this may be due to thecytotoxicity and enterotoxicity of C. difficile Toxin A as two separateand distinct biological functions.

Example 15 In Vivo Neutralization of C. Difficile Toxin A by AntibodiesAgainst Recombinant Toxin A Polypeptides

The ability of avian antibodies directed against the recombinant C.difficile toxin A fragment 1870-2680 (as expressed by pMA1870-2680; seeExample 13) to neutralize the enterotoxic activity of toxin A wasdemonstrated in Example 14. The ability of avian antibodies (IgYs)directed against other recombinant toxin A epitopes to neutralize nativetoxin A in vivo was next evaluated. This example involved: (a) thepreparation of IgYs against recombinant toxin A polypeptides; (b) invivo protection of hamsters against toxin A by treatment withanti-recombinant toxin A IgYs and (c) quantification of specificantibody concentration in CTA and Interval 6 IgY PEG preparations.

The nucleotide sequence of the coding region of the entire toxin Aprotein is listed in SEQ ID NO:5. The amino acid sequence of the entiretoxin A protein is listed in SEQ ID NO:6. The amino acid sequenceconsisting of amino acid residues 1870 through 2680 of toxin A is listedin SEQ ID NO:7. The amino acid sequence consisting of amino acidresidues 1870 through 1960 of toxin A is listed in SEQ ID NO:8.

a) Preparation of IgY's Against Recombinant Toxin A Polypeptides

Eggs were collected from Leghorn hens which have been immunized withrecombinant C. difficile toxin A polypeptide fragments encompassing theentire toxin A protein. The polypeptide fragments used as immunogenswere: 1) pMA 1870-2680 (Interval 6), 2) pPA 1100-1450 (Interval 4), and3) a mixture of fragments consisting of pMA 30-300 (Interval 1), pMA300-660 (Interval 2), pMA 660-1100 (Interval 3) and pMA 1450-1870(Interval 5). This mixture of immunogens is referred to as Interval1235. The location of each interval within the toxin A molecule is shownin FIG. 15A. In FIG. 15A, the following abbreviations are used: pPrefers to the pET23 vector (New England BioLabs); pM refers to thepMAL™-c vector (New England BioLabs); A refers to toxin A; the numbersrefer to the amino acid interval expressed in the clone. (For example,the designation pMA30-300 indicates that the recombinant clone encodesamino acids 30-300 of toxin A and the vector used was pMAL™-c).

The recombinant proteins were generated as described in Example 11. TheIgYs were extracted and solubilized in 0.1M carbonate buffer pH 9.5 fororal administration as described in Example 14(a). The IgY reactivitiesagainst each individual recombinant interval was evaluated by ELISA asdescribed in Example 13(c).

b) In Vivo Protection of Hamsters Against Toxin A By Treatment withAnti-Recombinant Toxin A Antibodies

The ability of antibodies raised against recombinant toxin Apolypeptides to provide in vivo protection against the enterotoxicactivity of toxin A was examined in the hamster model system. This assaywas performed as described in Example 14(b). Briefly, for each 40-50gram female Golden Syrian hamster (Charles River), 1 ml of IgY 4× (i.e.,resuspended in ¼ of the original yolk volume) PEG prep against Interval6, Interval 4 or Interval 1235 was mixed with 30 μg (LD₁₀₀ oral dose) ofC. difficile toxin A (Tech Lab). Preimmune IgY mixed with toxin A servedas a negative control. Antibodies raised against C. difficile toxoid A(Example 8) mixed with toxin A (CTA) served as a positive control. Themixture was incubated for 1 hour at 37° C. then orally administered tolightly etherized hamsters using an 18 G feeding needle. The animalswere then observed for the onset of diarrhea and death for a period ofapproximately 24 hours. The results are shown in Table 18. TABLE 18Study Outcome After 24 Hours Treatment group Healthy¹ Diarrhea² Dead³Preimmune 0 0 7 CTA 5 0 0 Interval 6 6 1 0 Interval 4 0 1 6 Interval1235 0 0 7¹Animal shows no sign of illness.²Animal developed diarrhea, but did not die.³Animal developed diarrhea and died.

Pre-treatment of toxin A with IgYs against Interval 6 prevented diarrheain 6 of 7 hamsters and completely prevented death in all 7. In contrast,as with preimmune IgY, IgYs against Interval 4 and Interval 1235 had noeffect on the onset of diarrhea and death in the hamsters.

c) Quantification of Specific Antibody Concentration in CTA and Interval6 IgY PEG Preparations

To determine the purity of IgY PEG preparations, an aliquot of apMA1870-2680 (Interval 6) IgY PEG preparation was chromatographed usingHPLC and a KW-803 sizing column (Shodex). The resulting profile ofabsorbance at 280 nm is shown in FIG. 16. The single large peakcorresponds to the predicted MW of IgY. Integration of the area underthe single large peak showed that greater than 95% of the protein elutedfrom the column was present in this single peak. This resultdemonstrated that the majority (>95%) of the material absorbing at 280nm in the PEG preparation corresponds to IgY. Therefore, absorbance at280 nm can be used to determine the total antibody concentration in PEGpreparations.

To determine the concentration of Interval 6-specific antibodies(expressed as percent of total antibody) within the CTA and pMA1870-2680(Interval 6) PEG preparations, defined quantities of these antibodypreparations were affinity purified on a pPA1870-2680(H) (shownschematically in FIG. 15B) affinity column and the specific antibodieswere quantified. In FIG. 15B the following abbreviations are used: pPrefers to the pET23 vector (New England BioLabs); pM refers to thepMAL™-c vector (New England BioLabs); pG refers to the pGEX vector(Pharmacia); pB refers to the PinPoint™ Xa vector (Promega); A refers totoxin A; the numbers refer to the amino acid interval expressed in theclone. The solid black ovals represent the MBP; the hatched ovalsrepresent glutathione S-transferase; the hatched circles represent thebiotin tag; and HHH represents the poly-histidine tag.

An affinity column containing recombinant toxin A repeat protein wasmade as follows. Four ml of PBS-washed Actigel resin (Sterogene) wascoupled with 5-10 mg of pPA1870-2680 inclusion body protein [prepared asdescribed in Example (17) and dialyzed into PBS] in a 15 ml tube(Falcon) containing 1/10 final volume Ald-coupling solution (1 M sodiumcyanoborohydride). Aliquots of the supernatant from the couplingreactions, before and after coupling, were assessed by Coomassiestaining of 7.5% SDS-PAGE gels. Based upon protein band intensities,greater than 6 mg of recombinant protein was coupled to the resin. Theresin was poured into a 10 ml column (BioRad), washed extensively withPBS, pre-eluted with 4 M guanidine-HCl (in 10 mM Tris-HCl, pH 8.0;0.005% thimerosal) and re-equilibrated with PBS. The column was storedat 4° C.

Aliquots of a pMA1870-2680 (Interval 6) or a CTA IgY polyclonal antibodypreparation (PEG prep) were affinity purified on the above affinitycolumn as follows. The column was attached to an UV monitor (ISCO) andwashed with PBS. For pMA1870-2680 IgY purification, a 2×PEG prep (filtersterilized using a 0.45μ filter; approximately 500 mg total IgY) wasapplied. The column was washed with PBS until the baseline wasre-established (the column flow-through was saved), washed with BBSTweento elute nonspecifically binding antibodies and re-equilibrated withPBS. Bound antibody was eluted from the column in 4 M guanidine-HCl (in10 mM Tris-HCl, pH 8.0; 0.005% thimerosal). The entire elution peak wascollected in a 15 ml tube (Falcon). The column was re-equilibrated andthe column eluate was re-chromatographed as described above. Theantibody preparation was quantified by UV absorbance (the elution bufferwas used to zero the spectrophotometer). Total purified antibody wasapproximately 9 mg and 1 mg from the first and second chromatographypasses, respectively. The low yield from the second pass indicated thatmost specific antibodies were removed by the first round ofchromatography. The estimated percentage of Interval 6 specificantibodies in the pMA1870-2680 PEG prep is approximately 2%.

The percentage of Interval 6 specific antibodies in the CTA PEG prep wasdetermined (utilizing the same column and methodology described above)to be approximately 0.5% of total IgY.

A 4×PEG prep contains approximately 20 mg/ml IgY. Thus in b) above,approximately 400 μg specific antibody in the Interval 6 PEG prepneutralized 30 μg toxin A in vivo.

Example 16 In Vivo Treatment of C. difficile Disease in Hamsters byRecombinant Interval 6 Antibodies

The ability of antibodies directed against recombinant Interval 6 oftoxin A to protect hamsters in vivo from C. difficile disease wasexamined. This example involved: (a) prophylactic treatment of C.difficile disease and (b) therapeutic treatment of C. difficile disease.

a) Prophylactic Treatment of C. difficile Disease

This experiment was performed as described in Example 9(b). Three groupseach consisting of 7 female 100 gram Syrian hamsters (Charles River)were prophylactically treated with either preimmune IgYs, IgYs againstnative toxin A and B [CTAB; see Example 8 (a) and (b)] or IgYs againstInterval 6. IgYs were prepared as 4×PEG preparations as described inExample 9(a).

The animals were orally dosed 3 times daily, roughly at 4 hourintervals, for 12 days with 1 ml antibody preparations diluted inEnsure®. Using estimates of specific antibody concentration from Example15(c), each dose of the Interval 6 antibody prep contained approximately400 μg of specific antibody. On day 2 each hamster was predisposed to C.difficile infection by the oral administration of 3.0 mg ofClindamycin-HCl (Sigma) in 1 ml of water. On day 3 the hamsters wereorally challenged with 1 ml of C. difficile inoculum strain ATCC 43596in sterile saline containing approximately 100 organisms. The animalswere then observed for the onset of diarrhea and subsequent death duringthe treatment period. The results are shown in Table 19. TABLE 19Lethality After 12 Days Of Treatment Treatment Number Animals NumberAnimals Group Alive Dead Preimmune 0 7 CTAB 6 1 Interval 6 7 0

Treatment of hamsters with orally-administered IgYs against Interval 6successfully protected 7 out of 7 (100%) of the animals from C.difficile disease. One of the hamsters in this group presented withdiarrhea which subsequently resolved during the course of treatment. Asshown previously in Example 9, antibodies to native toxin A and toxin Bwere highly protective. In this Example, 6 out of 7 animals survived inthe CTAB treatment group. All of the hamsters treated with preimmunesera came down with diarrhea and died. The survivors in both the CTABand Interval 6 groups remained healthy throughout a 12 daypost-treatment period. In particular, 6 out of 7 Interval 6-treatedhamsters survived at least 2 weeks after termination of treatment whichsuggests that these antibodies provide a long-lasting cure. Theseresults represent the first demonstration that antibodies generatedagainst a recombinant region of toxin A can prevent CDAD whenadministered passively to animals. These results also indicate thatantibodies raised against Interval 6 alone may be sufficient to protectanimals from C. difficile disease when administered prophylactically.

Previously others had raised antibodies against toxin A by activelyimmunizing hamsters against a recombinant polypeptide located within theInterval 6 region [Lyerly, D. M., et al. (1990) Curr. Microbiol. 21:29].FIG. 17 shows schematically the location of the Lyerly, et alintra-Interval 6 recombinant protein (cloned into the pUC vector) incomparison with the complete Interval 6 construct (pMA1870-2680) usedherein to generate neutralizing antibodies directed against toxin A. InFIG. 17, the solid black oval represents the MBP which is fused to thetoxin A Interval 6 in pMA1870-2680.

The Lyerly, et al. antibodies (intra-Interval 6) were only able topartially protect hamsters against C. difficile infection in terms ofsurvival (4 out of 8 animals survived) and furthermore, these antibodiesdid not prevent diarrhea in any of the animals. Additionally, animalstreated with the intra-Interval 6 antibodies [Lyerly, et al. (1990),supra] died when treatment was removed.

In contrast, the experiment shown above demonstrates that passiveadministration of anti-Interval 6 antibodies prevented diarrhea in 6 outof 7 animals and completely prevented death due to CDAD. Furthermore, asdiscussed above, passive administration of the anti-Interval 6antibodies provides a long lasting cure (i.e., treatment could bewithdrawn without incident).

b) Therapeutic Treatment of C. difficile Disease: In Vivo Treatment ofan Established C. difficile Infection in Hamsters with RecombinantInterval 6 Antibodies

The ability of antibodies against recombinant interval 6 of toxin A totherapeutically treat C. difficile disease was examined. The experimentwas performed essentially as described in Example 10(b). Three groups,each containing seven to eight female Golden Syrian hamsters (100 geach; Charles River) were treated with either preimmune IgY, IgYsagainst native toxin A and toxin B (CTAB) and IgYs against Interval 6.The antibodies were prepared as described above as 4×PEG preparations.

The hamsters were first predisposed to C. difficile infection with a 3mg dose of Clindamycin-HCl (Sigma) administered orally in 1 ml of water.Approximately 24 hrs later, the animals were orally challenged with 1 mlof C. difficile strain ATCC 43596 in sterile saline containingapproximately 200 organisms. One day after infection, the presence oftoxin A and B was determined in the feces of the hamsters using acommercial immunoassay kit (Cytoclone A+B EPA, Cambridge Biotech) toverify establishment of infection. Four members of each group wererandomly selected and tested. Feces from an uninfected hamster wastested as a negative control. All infected animals tested positive forthe presence of toxin according to the manufacturer's procedure. Theinitiation of treatment then started approximately 24 hr post-infection.

The animals were dosed daily at roughly 4 hr intervals with 1 mlantibody preparation diluted in Ensure® (Ross Labs). The amount ofspecific antibodies given per dose (determined by affinity purification)was estimated to be about 400 μg of anti-interval 6 IgY (for animals inthe Interval 6 group) and 100 μg and 70 μg of anti-toxin A (Interval6-specific) and anti-toxin B (Interval 3-specific; see Example 19),respectively, for the CTAB preparation. The animals were treated for 9days and then observed for an additional 4 days for the presence ofdiarrhea and death. The results indicating the number of survivors andthe number of dead 4 days post-infection are shown in Table 20. TABLE 20In vivo Therapeutic Treatment With Interval 6 Antibodies TreatmentNumber Animals Number Animals Group Alive Dead Preimmune 4 3 CTAB 8 0Interval 8 0

Antibodies directed against both Interval 6 and CTAB successfullyprevented death from C. difficile when therapeutically administered 24hr after infection. This result is significant since many investigatorsbegin therapeutic treatment of hamsters with existing drugs (e.g.,vancomycin, phenelfamycins, tiacumicins, etc.) 8 hr post-infection[Swanson, et al. (1991) Antimicrobial Agents and Chemotherapy 35:1108and (1989) J. Antibiotics 42:94].

Forty-two percent of hamsters treated with preimmune IgY died from CDAD.While the anti-Interval 6 antibodies prevented death in the treatedhamsters, they did not eliminate all symptoms of CDAD as 3 animalspresented with slight diarrhea. In addition, one CTAB-treated and onepreimmune-treated animal also had diarrhea 14 days post-infection. Theseresults indicate that anti-Interval 6 antibodies provide an effectivemeans of therapy for CDAD.

Example 17 Induction of Toxin A Neutralizing Antibodies Requires SolubleInterval 6 Protein

As shown in Examples 11(d) and 15, expression of recombinant proteins inE. coli may result in the production of either soluble or insolubleprotein. If insoluble protein is produced, the recombinant protein issolubilized prior to immunization of animals. To determine whether, oneor both of the soluble or insoluble recombinant proteins could be usedto generate neutralizing antibodies to toxin A, the following experimentwas performed. This example involved a) expression of the toxin Arepeats and subfragments of these repeats in E. coli using a variety ofexpression vectors; b) identification of recombinant toxin A repeats andsub-regions to which neutralizing antibodies bind; and c) determinationof the neutralization ability of antibodies raised against soluble andinsoluble toxin A repeat immunogen.

a) Expression of the Toxin A Repeats and Subfragments of these Repeatsin E. coli Using a Variety of Expression Vectors

The Interval 6 immunogen utilized in Examples 15 and 16 was thepMA1870-2680 protein, in which the toxin A repeats are expressed as asoluble fusion protein with the MBP (described in Example 11).Interestingly, expression of this region (from the SpeI site to the endof the repeats, see FIG. 15B) in three other expression constructs, aseither native (pPA1870-2680), poly-His tagged [pPA1870-2680 (H)] orbiotin-tagged (pBA1870-2680) proteins resulted in completely insolubleprotein upon induction of the bacterial host (see FIG. 15B). The hoststrain BL21 (Novagen) was used for expression of pBA1870-2680 and hoststrain BL21(DE3) (Novagen) was used for expression of pPA1870-2680 andpPA1870-2680(H). These insoluble proteins accumulated to high levels ininclusion bodies. Expression of recombinant plasmids in E. coli hostcells grown in 2×YT medium was performed as described [Williams, et al(1995), supra].

As summarized in FIG. 15B, expression of fragments of the toxin Arepeats (as either N-terminal SpeI-EcoRI fragments, or C-terminalEcoRI-end fragments) also yielded high levels of insoluble protein usingpGEX (pGA1870-2190), PinPoint™-Xa (pBA1870-2190 and pBA2250-2680) andpET expression systems (pPA1870-2190). The pGEX and pET expressionsystems are described in Example 11. The PinPoint™-Xa expression systemdrives the expression of fusion proteins in E. coli. Fusion proteinsfrom PinPoint™-Xa vectors contain a biotin tag at the amino-terminal endand can be affinity purified SoftLink™ Soft Release avidin resin(Promega) under mild denaturing conditions (5 mM biotin).

The solubility of expressed proteins from the pPG1870-2190 andpPA1870-2190 expression constructs was determined after induction ofrecombinant protein expression under conditions reported to enhanceprotein solubility [These conditions comprise growth of the host atreduced temperature (30° C.) and the utilization of high (1 mM IPTG) orlow (0.1 mM IPTG) concentrations of inducer [Williams et al. (1995),supra]. All expressed recombinant toxin A protein was insoluble underthese conditions. Thus, expression of these fragments of the toxin Arepeats in pET and PGEX expression vectors results in the production ofinsoluble recombinant protein even when the host cells are grown atreduced temperature and using lower concentrations of the inducer.Although expression of these fragments in pMa1 vectors yielded affinitypurifiable soluble fusion protein, the protein was either predominantlyinsoluble (pMA1870-2190) or unstable (pMA2250-2650). Attempts tosolubilize expressed protein from the pMA1870-2190 expression constructusing reduced temperature or lower inducer concentration (as describedabove) did not improve fusion protein solubility.

Collectively, these results demonstrate that expression of the toxin Arepeat region in E. coli results in the production of insolublerecombinant protein, when expressed as either large (aa 1870-2680) orsmall (aa 1870-2190 or aa 2250-2680) fragments, in a variety ofexpression vectors (native or poly-His tagged pET, pGEX or PinPoint™-Xavectors), utilizing growth conditions shown to enhance proteinsolubility. The exception to this rule were fusions with the MBP, whichenhanced protein solubility, either partially (pMA1870-2190) or fully(pMA1870-2680).

B) Identification of Recombinant Toxin A Repeats and Sub-Regions towhich Neutralizing Antibodies Bind

Toxin A repeat regions to which neutralizing antibodies bind wereidentified by utilizing recombinant toxin A repeat region proteinsexpressed as soluble or insoluble proteins to deplete protectiveantibodies from a polyclonal pool of antibodies against native C.difficile toxin A. An in vivo assay was developed to evaluate proteinsfor the ability to bind neutralizing antibodies.

The rational for this assay is as follows. Recombinant proteins werefirst pre-mixed with antibodies against native toxin A (CTA antibody;generated in Example 8) and allowed to react. Subsequently, C. difficiletoxin A was added at a concentration lethal to hamsters and the mixturewas administered to hamsters via IP injection. If the recombinantprotein contains neutralizing epitopes, the CTA antibodies would losetheir ability to bind toxin A resulting in diarrhea and/or death of thehamsters.

The assay was performed as follows. The lethal dose of toxin A whendelivered orally to nine 40 to 50 g Golden Syrian hamsters (Sasco) wasdetermined to be 10 to 30 μg. The PEG-purified CTA antibody preparationwas diluted to 0.5× concentration (i.e., the antibodies were diluted attwice the original yolk volume) in 0.1 M carbonate buffer, pH 9.5. Theantibodies were diluted in carbonate buffer to protect them from aciddegradation in the stomach. The concentration of 0.5× was used becauseit was found to be the lowest effective concentration against toxin A.The concentration of Interval 6-specific antibodies in the 0.5×CTA prepwas estimated to be 10-15 μg/ml (estimated using the method described inExample 15).

The inclusion body preparation [insoluble Interval 6 protein;pPA1870-2680(H)] and the soluble Interval 6 protein [pMA1870-2680; seeFIG. 15] were both compared for their ability to bind to neutralizingantibodies against C. difficile toxin A (CTA). Specifically, 1 to 2 mgof recombinant protein was mixed with 5 ml of a 0.5×CTA antibody prep(estimated to contain 60-70 μg of Interval 6-specific antibody). Afterincubation for 1 hr at 37° C., CTA (Tech Lab) at a final concentrationof 30 μg/ml was added and incubated for another 1 hr at 37° C. One ml ofthis mixture containing 30 μg of toxin A (and 10-15 μg of Interval⁴6-specific antibody) was administered orally to 40-50 g Golden Syrianhamsters (Sasco). Recombinant proteins that result in the loss ofneutralizing capacity of the CTA antibody would indicate that thoseproteins contain neutralizing epitopes. Preimmune and CTA antibodies(both at 0.5×) without the addition of any recombinant protein served asnegative and positive controls, respectively.

Two other inclusion body preparations, both expressed as insolubleproducts in the pET vector, were tested; one containing a differentinsert (toxin B fragment) other than Interval 6 called pPB1850-2070 (seeFIG. 18) which serves as a control for insoluble Interval 6, the otherwas a truncated version of the Interval 6 region called pPA1870-2190(see FIG. 15B). The results of this experiment are shown in Table 21.TABLE 21 Binding Of Neutralizing Antibodies By Soluble Interval 6Protein Study Outcome After 24 Hours Treatment Group¹ Healthy² Diarrhea³Dead⁴ Preimmune Ab 0 3 2 CTA Ab 4 1 0 CTA Ab + Int 6 1 2 2 (soluble) CTAAb + Int 6 5 0 0 (insoluble) CTA Ab + pPB1850-2070 5 0 0 CTA Ab +pPA1870-2190 5 0 0¹ C. difficile toxin A (CTA) was added to each group.²Animals showed no signs of illness.³Animals developed diarrhea but did not die.⁴Animals developed diarrhea and died.

Preimmune antibody was ineffective against toxin A, while anti-CTAantibodies at a dilute 0.5× concentration almost completely protectedthe hamsters against the enterotoxic effects of CTA. The addition ofrecombinant proteins pPB1850-2070 or pPA1870-2190 to the anti-CTAantibody had no effect upon its protective ability, indicating thatthese recombinant proteins do not bind to neutralizing antibodies. Onthe other hand, recombinant Interval 6 protein was able to bind toneutralizing anti-CTA antibodies and neutralized the in vivo protectiveeffect of the anti-CTA antibodies. Four out of five animals in the grouptreated with anti-CTA antibodies mixed with soluble Interval 6 proteinexhibited toxin associated toxicity (diarrhea and death). Moreover, theresults showed that Interval 6 protein must be expressed as a solubleproduct in order for it to bind to neutralizing anti-CTA antibodiessince the addition of insoluble Interval 6 protein had no effect on theneutralizing capacity of the CTA antibody prep.

c) Determination of Neutralization Ability of Antibodies Raised AgainstSoluble and Insoluble Toxin A Repeat Immunogen

To determine if neutralizing antibodies are induced against solubilizedinclusion bodies, insoluble toxin A repeat protein was solubilized andspecific antibodies were raised in chickens. Insoluble pPA1870-2680protein was solubilized using the method described in Williams et al.(1995), supra. Briefly, induced cultures (500 ml) were pelleted bycentrifugation at 3,000×g for 10 min at 4° C. The cell pellets wereresuspended thoroughly in 10 ml of inclusion body sonication buffer (25mM HEPES pH 7.7, 100 mM KCl, 12.5 mM MgCl₂, 20% glycerol, 0.1% (v/v)Nonidet P-40, 1 mM DTT). The suspension was transferred to a 30 mlnon-glass centrifuge tube. Five hundred μl of 10 mg/ml lysozyme wasadded and the tubes were incubated on ice for 30 min. The suspension wasthen frozen at −70° C. for at least 1 hr. The suspension was thawedrapidly in a water bath at room temperature and then placed on ice. Thesuspension was then sonicated using at least eight 15 sec bursts of themicroprobe (Branson Sonicator Model No. 450) with intermittent coolingon ice.

The sonicated suspension was transferred to a 35 ml Oakridge tube andcentrifuged at 6,000×g for 10 min at 4° C. to pellet the inclusionbodies. The pellet was washed 2 times by pipetting or vortexing infresh, ice-cold RIPA buffer [0.1% SDS, 1% Triton X-100, 1% sodiumdeoxycholate in TBS (25 mM Tris-Cl pH 7.5, 150 mM NaCl)]. The inclusionbodies were recentrifuged after each wash. The inclusion bodies weredried and transferred using a small metal spatula to a 15 ml tube(Falcon). One ml of 10% SDS was added and the pellet was solubilized bygently pipetting the solution up and down using a 1 ml micropipettor.The solubilization was facilitated by heating the sample to 95° C. whennecessary.

Once the inclusion bodies were in solution, the samples were dilutedwith 9 volumes of PBS. The protein solutions were dialyzed overnightagainst a 100-fold volume of PBS containing 0.05% SDS at roomtemperature. The dialysis buffer was then changed to PBS containing0.01% SDS and the samples were dialyzed for several hours to overnightat room temperature. The samples were stored at 4° C. until used. Priorto further use, the samples were warmed to room temperature to allow anyprecipitated SDS to go back into solution.

The inclusion body preparation was used to immunize hens. The proteinwas dialyzed into PBS and emulsified with approximately equal volumes ofCFA for the initial immunization or IFA for subsequent boosterimmunizations. On day zero, for each of the recombinant preparations,two egg laying white Leghorn hens were each injected at multiple sites(IM and SC) with 1 ml of recombinant protein-adjuvant mixture containingapproximately 0.5-1.5 mg of recombinant protein. Booster immunizationsof 1.0 mg were given of days 14 and day 28. Eggs were collected on day32 and the antibody isolated using PEG as described in Example 14(a).High titers of toxin A specific antibodies were present (as assayed byELISA, using the method described in Example 13). Titers were determinedfor both antibodies against recombinant polypeptides pPA1870-2680 andpMA1870-2680 and were found to be comparable at >1:62,500.

Antibodies against soluble Interval 6 (pMA1870-2680) and insolubleInterval 6 [(inclusion body), pPA1870-2680] were tested for neutralizingability against toxin A using the in vivo assay described in Example15(b). Preimmune antibodies and antibodies against toxin A (CTA) servedas negative and positive controls, respectively. The results are shownin Table 22. TABLE 22 Neutralization Of Toxin A By Antibodies AgainstSoluble Interval 6 Protein Study Outcome After 24 Hours AntibodyTreatment Group Healthy¹ Diarrhea² Dead³ Preimmune 1 0 4 CTA 5 0 0Interval 6 5 0 0 (Soluble)⁴ Interval 6 0 2 3 (Insoluble)¹Animals showed no sign of illness.²Animal developed diarrhea but did not die.³Animal developed diarrhea and died.⁴400 μg/ml.

Antibodies raised against native toxin A were protective while preimmuneantibodies had little effect. As found using the in vitro CHO assay[described in Example 8(d)] where antibodies raised against the solubleInterval 6 could partially neutralize the effects of toxin A, here theywere able to completely neutralize toxin A in vivo. In contrast, theantibodies raised against the insoluble Interval 6 was unable toneutralize the effects of toxin A in vivo as shown above (Table 22) andin vitro as shown in the CHO assay [described in Example 8(d)].

These results demonstrate that soluble toxin A repeat immunogen isnecessary to induce the production of neutralizing antibodies inchickens, and that the generation of such soluble immunogen is obtainedonly with a specific expression vector (pMa1) containing the toxin Aregion spanning aa 1870-2680. That is to say, insoluble protein that issubsequently solubilized does not result in a toxin A antigen that willelicit a neutralizing antibody.

Example 18 Cloning and Expression of the C. difficile Toxin B Gene

The toxin B gene has been cloned and sequenced; the amino acid sequencededuced from the cloned nucleotide sequence predicts a MW of 269.7 kDfor toxin B [Barroso et al., Nucl. Acids Res. 18:4004 (1990)]. Thenucleotide sequence of the coding region of the entire toxin B gene islisted in SEQ ID NO:9. The amino acid sequence of the entire toxin Bprotein is listed in SEQ ID NO:10 The amino acid sequence consisting ofamino acid residues 1850 through 2360 of toxin B is listed in SEQ IDNO:11. The amino acid sequence consisting of amino acid residues 1750through 2360 of toxin B is listed in SEQ ID NO:12.

Given the expense and difficulty of isolating native toxin B protein, itwould be advantageous to use simple and inexpensive procaryoticexpression systems to produce and purify high levels of recombinanttoxin B protein for immunization purposes. Ideally, the isolatedrecombinant protein would be soluble in order to preserve nativeantigenicity, since solubilized inclusion body proteins often do notfold into native conformations. Indeed as shown in Example 17,neutralizing antibodies against recombinant toxin A were only obtainedwhen soluble recombinant toxin A polypeptides were used as theimmunogen. To allow ease of purification, the recombinant protein shouldbe expressed to levels greater than 1 mg/liter of E. coli culture.

To determine whether high levels of recombinant toxin B protein could beproduced in E. coli, fragments of the toxin B gene were cloned intovarious prokaryotic expression vectors, and assessed for the ability toexpress recombinant toxin B protein in E. coli. This Example involved(a) cloning of the toxin B gene and (b) expression of the toxin B genein E. coli.

a) Cloning of the Toxin B Gene

The toxin B gene was cloned using PCR amplification from C. difficilegenomic DNA. Initially, the gene was cloned in two overlappingfragments, using primer pairs P5/P6 and P7/P8. The location of theseprimers along the toxin B gene is shown schematically in FIG. 18. Thesequence of each of these primers is:

P5: 5′ TAGAAAAAATGGCAAATGT 3′; (SEQ ID NO:11) P6: 5′TTTCATCTTGTAGAGTCAAAG 3′; (SEQ ID NO:12) P7: 5′ GATGCCACAAGATGATTTAGTG3′; (SEQ ID NO:13) and P8: 5′ CTAATTGAGCTGTATCAGGATC 3′. (SEQ ID NO:14)

FIG. 18 also shows the location of the following primers along the toxinB gene: P9 which consists of the sequence 5′ CGGAATTCCTAGAAAAAATGGCAAATG3′ (SEQ ID NO:15); P10 which consists of the sequence 5′ GCTCTAGAATGACCATAAGCTAGCCA 3′ (SEQ ID NO:16); P11 which consists of the sequence 5′CGGAATTCGAGTTGGTAGAAAGGTGGA 3′ (SEQ ID NO: 17); P13 which consists ofthe sequence 5′ CGGAATTCGGTTATTATCTTAAGGATG 3′ (SEQ ID NO:18); and P14which consists of the sequence 5′ CGGAATTCTTGATAACTGGAT TTGTGAC 3′ (SEQID NO:19). The amino acid sequence consisting of amino acid residues1852 through 2362 of toxin B is listed in SEQ ID NO:20. The amino acidsequence consisting of amino acid residues 1755 through 2362 of toxin Bis listed in SEQ ID NO:21.

Clostridium difficile VPI strain 10463 was obtained from the AmericanType Culture Collection (ATCC 43255) and grown under anaerobicconditions in brain-heart infusion medium (Becton Dickinson). Highmolecular-weight C. difficile DNA was isolated essentially as described[Wren and Tabaqchali (1987) J. Clin. Microbiol., 25:2402], except 1) 100μg/ml proteinase K in 0.5% SDS was used to disrupt the bacteria and 2)cetyltrimethylammonium bromide (CTAB) precipitation [as described byAusubel et al, Eds., Current Protocols in Molecular Biology, Vol. 2(1989) Current Protocols] was used to remove carbohydrates from thecleared lysate. Briefly, after disruption of the bacteria withproteinase K and SDS, the solution is adjusted to approximately 0.7 MNaCl by the addition of a 1/7 volume of 5M NaCl. A 1/10 volume ofCTAB/NaCl (10% CTAB in 0.7 M NaCl) solution was added and the solutionwas mixed thoroughly and incubated 10 min at 65° C. An equal volume ofchloroform/isoamyl alcohol (24:1) was added and the phases werethoroughly mixed. The organic and aqueous phases were separated bycentrifugation in a microfuge for 5 min. The aqueous supernatant wasremoved and extracted with phenol/chloroform/isoamyl alcohol (25:24:1).The phases were separated by centrifugation in a microfuge for 5 min.The supernatant was transferred to a fresh tube and the DNA wasprecipitated with isopropanol. The DNA precipitate was pelleted by briefcentrifugation in a microfuge. The DNA pellet was washed with 70%ethanol to remove residual CTAB. The DNA pellet was then dried andredissolved in TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Theintegrity and yield of genomic DNA was assessed by comparison with aserial dilution of uncut lambda DNA after electrophoresis on an agarosegel.

Toxin B fragments were cloned by PCR utilizing a proofreadingthermostable DNA polymerase [native Pfu polymerase (Stratagene)]. Thehigh fidelity of this polymerase reduces the mutation problemsassociated with amplification by error prone polymerases (e.g., Taqpolymerase). PCR amplification was performed using the PCR primer pairsP5 (SEQ ID NO:11) with P6 (SEQ ID NO:12) and P7 (SEQ ID NO: 13) with P8(SEQ ID NO:14) in 50 μl reactions containing 10 mM Tris-HCl pH 8.3, 50mM KCl, 1.5 mM MgCl₂, 200 μM of each dNTP, 0.2 μM each primer, and 50 ngC. difficile genomic DNA. Reactions were overlaid with 100 μl mineraloil, heated to 94° C. for 4 min, 0.5 μl native Pfu polymerase(Stratagene) was added, and the reactions were cycled 30 times at 94° C.for 1 min, 50° C. for 1 min, 72° C. (2 min for each kb of sequence to beamplified), followed by 10 min at 72° C. Duplicate reactions werepooled, chloroform extracted, and ethanol precipitated. After washing in70% ethanol, the pellets were resuspended in 50 μl TE buffer (10 mMTris-HCl pH 8.0, 1 mM EDTA).

The P5/P6 amplification product was cloned into pUC19 as outlined below.10 μl aliquots of DNA were gel purified using the Prep-a-Gene kit(BioRad), and ligated to SmaI restricted pUC19 vector. Recombinantclones were isolated and confirmed by restriction digestion usingstandard recombinant molecular biology techniques (Sambrook et al.,1989). Inserts from two independent isolates were identified in whichthe toxin B insert was oriented such that the vector BamHI and SacIsites were 5′ and 3′ oriented, respectively (pUCB10-1530). Theinsert-containing BamHI/SacI fragment was cloned into similarly cutpET23a-c vector DNA, and protein expression was induced in small scalecultures (5 ml) of identified clones.

Total protein extracts were isolated, resolved on SDS-PAGE gels, andtoxin B protein identified by Western analysis utilizing a goatanti-toxin B affinity purified antibody (Tech Lab). Procedures forprotein induction, SDS-PAGE, and Western blot analysis were performed asdescribed in Williams et al. (1995), supra. In brief, 5 ml cultures ofbacteria grown in 2XYT containing 100 μg/ml ampicillin containing theappropriate recombinant clone were induced to express recombinantprotein by addition of IPTG to 1 mM. The E. coli hosts used were:BL21(DE3) or BL21(DE3)LysS (Novagen) for pET plasmids.

Cultures were induced by the addition of IPTG to a final concentrationof 1.0 mM when the cell density reached 0.5 OD₆₀₀, and induced proteinwas allowed to accumulate for two hrs after induction. Protein sampleswere prepared by pelleting 1 ml aliquots of bacteria by centrifugation(1 min in microfuge), and resuspension of the pelleted bacteria in 150μl of 2×SDS-PAGE sample buffer (0.125 mM Tris-HCl pH 6.8, 2 mM EDTA, 6%SDS, 20% glycerol, 0.025% bromophenol blue; β-mercaptoethanol is addedto 5% before use). The samples were heated to 95° C. for 5 min, thencooled and 5 or 10 μls loaded on 7.5% SDS-PAGE gels. High molecularweight protein markers (BioRad) were also loaded, to allow estimation ofthe MW of identified fusion proteins. After electrophoresis, protein wasdetected either generally by staining the gels with Coomassie Blue, orspecifically, by blotting to nitrocellulose for Western blot detectionof specific immunoreactive protein. The MW of induced toxin B reactiveprotein allowed the integrity of the toxin B reading frame to bedetermined.

The pET23b recombinant (pPB10-1530) expressed high MW recombinant toxinB reactive protein, consistent with predicted values. This confirmedthat frame terminating errors had not occurred during PCR amplification.A pET23b expression clone containing the 10-1750aa interval of the toxinB gene was constructed, by fusion of the EcoRV-SpeI fragment of theP7/P8 amplification product to the P5-EcoRV interval of the P5/P6amplification product (see FIG. 18) in pPB10-1530. The integrity of thisclone (pPB10-1750) was confirmed by restriction mapping, and Westernblot detection of expressed recombinant toxin B protein. Levels ofinduced protein from both pPB10-1530 and pPB10-1750 were too low tofacilitate purification of usable amounts of recombinant toxin Bprotein. The remaining 1750-2360 aa interval was directly cloned intopMAL or pET expression vectors from the P7/P8 amplification product asdescribed below.

b) Expression of the Toxin B Gene

i) Overview of Expression Methodologies

Fragments of the toxin B gene were expressed as either native or fusionproteins in E. coli. Native proteins were expressed in either thepET23a-c or pET16b expression vectors (Novagen). The pET23 vectorscontain an extensive polylinker sequence in all three reading frames(a-c vectors) followed by a C-terminal poly-histidine repeat. The pET16bvector contains a N-terminal poly-histidine sequence immediately 5′ to asmall polylinker. The poly-histidine sequence binds to Ni-Chelatecolumns and allows affinity purification of tagged target proteins[Williams et al. (1995), supra]. These affinity tags are small (10 aafor pET16b, 6 aa for pET23) allowing the expression and affinitypurification of native proteins with only limited amounts of foreignsequences.

An N-terminal histidine-tagged derivative of pET16b containing anextensive cloning cassette was constructed to facilitate cloning ofN-terminal poly-histidine tagged toxin B expressing constructs. This wasaccomplished by replacement of the promoter region of the pET23a and bvectors with that of the pET16b expression vector. Each vector wasrestricted with BglII and NdeI, and the reactions resolved on a 1.2%agarose gel. The pET16b promoter region (contained in a 200 bpBglII-NdeI fragment) and the promoter-less pET23 a or b vectors were cutfrom the gel, mixed and Prep-A-Gene (BioRad) purified. The eluted DNAwas ligated, and transformants screened for promoter replacement by NcoIdigestion of purified plasmid DNA (the pET16b promoter contains thissite, the pET23 promoter does not). These clones (denoted pETHisa or b)contain the pET16b promoter (consisting of a pT7-lac promoter, ribosomebinding site and poly-histidine (10aa) sequence) fused at the NdeI siteto the extensive pET23 polylinker.

All MBP fusion proteins were constructed and expressed in the pMAL™-c orpMAL™-p2 vectors (New England Biolabs) in which the protein of interestis expressed as a C-terminal fusion with MBP. All pET plasmids wereexpressed in either the BL21(DE3) or BL21(DE3)LysS expression hosts,while pMa1 plasmids were expressed in the BL21 host.

Large scale (500 mls to 1 liter) cultures of each recombinant were grownin 2XYT broth, induced, and soluble protein fractions were isolated asdescribed [Williams, et al. (1995), supra]. The soluble protein extractswere affinity chromatographed to isolate recombinant fusion protein, asdescribed [Williams et al., (1995) supra]. In brief, extracts containingtagged pET fusions were chromatographed on a nickel chelate column, andeluted using imidazole salts or low pH (pH 4.0) as described by thedistributor (Novagen or Qiagen). Extracts containing soluble pMAL fusionprotein were prepared and chromatographed in PBS buffer over an amyloseresin (New England Biolabs) column, and eluted with PBS containing 10 mMmaltose as described [Williams et al. (1995), supra].

ii) Overview of Toxin B Expression

In both large expression constructs described in (a) above, only lowlevel (i.e., less than 1 mg/liter of intact or nondegraded recombinantprotein) expression of recombinant protein was detected. A number ofexpression constructs containing smaller fragments of the toxin B genewere then constructed, to determine if small regions of the gene can beexpressed to high levels (i.e., greater than 1 mg/liter intact protein)without extensive protein degradation. All were constructed by in framefusions of convenient toxin B restriction fragments to either thepMAL-c, pET23a-c, pET16b or pETHisa-b expression vectors, or byengineering restriction sites at specific locations using PCRamplification [using the same conditions described in (a) above]. In allcases, clones were verified by restriction mapping, and, whereindicated, DNA sequencing.

Protein preparations from induced cultures of each of these constructswere analyzed, by SDS-PAGE, to estimate protein stability (CoomassieBlue stairung) and immunoreactivity against anti-toxin B specificantiserum (Western analysis). Higher levels of intact (i.e.,nondegraded), full length fusion proteins were observed with the smallerconstructs as compared with the larger recombinants, and a series ofexpression constructs spanning the entire toxin B gene were constructed(FIGS. 18, 19 and 20 and Table 23).

Constructs that expressed significant levels of recombinant toxin Bprotein (greater than 1 mg/liter intact recombinant protein) in E. coliwere identified and a series of these clones that spans the toxin B geneare shown in FIG. 19 and summarized in Table 23. These clones wereutilized to isolate pure toxin B recombinant protein from the entiretoxin B gene. Significant protein yields were obtained from pMALexpression constructs spanning the entire toxin B gene, and yields offull length soluble fusion protein ranged from an estimated 1 mg/literculture (pMB1100-1530) to greater than 20 mg/liter culture(pMB1750-2360).

Representative purifications of MBP and poly-histidine-tagged toxin Brecombinants are shown in FIGS. 21 and 22. FIG. 21 shows a CoomassieBlue stained 7.5% SDS-PAGE gel on which various protein samplesextracted from bacteria harboring pMB1850-2360 were electrophoresed.Samples were loaded as follows: Lane 1: protein extracted from uninducedculture; Lane 2: induced culture protein; Lane 3: total protein frominduced culture after sonication; Lane 4: soluble protein; and Lane 5:eluted affinity purified protein. FIG. 22 depicts the purification ofrecombinant proteins expressed in bacteria harboring either pPB1850-2360(Lanes 1-3) or pPB1750-2360 (Lanes 4-6). Samples were loaded as follows:uninduced total protein (Lanes 1 and 4); induced total protein (Lanes 2and 5); and eluted affinity purified protein (Lanes 3 and 6). The broadrange molecular weight protein markers (BioRad) are shown in Lane 7.

Thus, although high level expression was not attained using largeexpression constructs from the toxin B gene, usable levels ofrecombinant protein were obtained by isolating induced protein from aseries of smaller PMAL expression constructs that span the entire toxinB gene.

These results represent the first demonstration of the feasibility ofexpressing recombinant toxin B protein to high levels in E. coli. Aswell, expression of small regions of the putative ligand binding domain(repeat region) of toxin B as native protein yielded insoluble protein,while large constructs, or fusions to MBP were soluble (FIG. 19),demonstrating that specific methodologies are necessary to producesoluble fusion protein from this interval.

iii) Clone Construction and Expression Details

A portion of the toxin B gene containing the toxin B repeat region[amino acid residues 1852-2362 of toxin B (SEQ ID NO:20)] was isolatedby PCR amplification of this interval of the toxin B gene from C.difficile genomic DNA. The sequence, and location within the toxin Bgene, of the two PCR primers [P7 (SEQ ID NO:13) and P8 (SEQ ID NO: 14)]used to amplify this region are shown in FIG. 18.

DNA from the PCR amplification was purified by chloroform extraction andethanol precipitation as described above. The DNA was restricted withSpeI, and the cleaved DNA was resolved by agarose gel electrophoresis.The restriction digestion with SpeI cleaved the 3.6 kb amplificationproduct into a 1.8 kb doublet band. This doublet band was cut from thegel and mixed with appropriately cut, gel purified pMALc or pET23bvector. These vectors were prepared by digestion with HindIII, fillingin the overhanging ends using the Klenow enzyme, and cleaving with XbaI(pMALc) or NheI (pET23b). The gel purified DNA fragments were purifiedusing the Prep-A-Gene kit (BioRad) and the DNA was ligated, transformedand putative recombinant clones analyzed by restriction mapping.

pET and pMa1 clones containing the toxin B repeat insert (aa interval1750-2360 of toxin B) were verified by restriction mapping, usingenzymes that cleaved specific sites within the toxin B region. In bothcases fusion of the toxin B SpeI site with either the compatible XbaIsite (pMa1) or compatible NheI site (pET) is predicted to create an inframe fusion. This was confirmed in the case of the pMB1750-2360 cloneby DNA sequencing of the clone junction and 5′ end of the toxin B insertusing a MBP specific primer (New England Biolabs). In the case of thepET construct, the fusion of the blunt ended toxin B 3′ end to thefilled HindIII site should create an in-frame fusion with the C-terminalpoly-histidine sequence in this vector. The pPB1750-2360 clone selectedhad lost, as predicted, the HindIII site at this clone junction; thiseliminated the possibility that an additional adenosine residue wasadded to the 3′ end of the PCR product by a terminal transferaseactivity of the Pfu polymerase, since fusion of this adenosine residueto the filled HindIII site would regenerate the restriction site (andwas observed in several clones).

One liter cultures of each expression construct were grown, and fusionprotein purified by affinity chromatography on either an amylose resincolumn (pMAL constructs; resin supplied by New England Biolabs) orNi-chelate column (pET constructs; resin supplied by Qiagen or Novagen)as described [Williams et al. (1995), supra]. The integrity and purityof the fusion proteins were determined by Coomassie staining of SDS-PAGEprotein gels as well as Western blot analysis with either an affinitypurified goat polyclonal antiserum (Tech Lab), or a chicken polyclonalPEG prep, raised against the toxin B protein (CTB) as described above inExample 8. In both cases, affinity purification resulted in yields inexcess of 20 mg protein per liter culture, of which greater than 90% wasestimated to be full-length recombinant protein. It should be noted thatthe poly-histidine affinity tagged protein was released from the QiagenNi-NTA resin at low imidazole concentration (60 mM), necessitating theuse of a 40 mM imidazole rather than a 60 mM imidazole wash step duringpurification.

A periplasmically secreted version of pMB1750-2360 was constructed byreplacement of the promoter and MBP coding region of this construct withthat from a related vector (pMAL™-p2; New England Biolabs) in which asignal sequence is present at the N-terminus of the MBP, such thatfusion protein is exported. This was accomplished by substituting aBglII-EcoRV promoter fragment from pMAL-p2 into pMB1750-2360. The yieldsof secreted, affinity purified protein (recovered from osmotic shockextracts as described by Riggs in Current Protocols in MolecularBiology, Vol. 2, Ausubel, et al., Eds. (1989), Current Protocols, pp.16.6.1-16.6.14] from this vector (pMBp1750-2360) were 6.5 mg/literculture, of which 50% was estimated to be full-length fusion protein.

The interval was also expressed in two non-overlapping fragments.pMB1750-1970 was constructed by introduction of a frameshift intopMB1750-2360, by restriction with HindIII, filling in the overhangingends and religation of the plasmid. Recombinant clones were selected byloss of the HindIII site, and further restriction map analysis.Recombinant protein expression from this vector was more than 20mg/liter of greater than 90% pure protein.

The complementary region was expressed in pMB1970-2360. This constructwas created by removal of the 1750-1970 interval of pMB1750-2360. Thiswas accomplished by restriction of this plasmid with EcoRI (in the pMa1cpolylinker 5′ to the insert) and III, filling in the overhanging ends,and religation of the plasmid. The resultant plasmid, pMB1970-2360, wasmade using both intracellularly and secreted versions of thepMB1750-2360 vector.

No fusion protein was secreted in the pMBp1970-2360 version, perhaps dueto a conformational constraint that prevents export of the fusionprotein. However, the intracellularly expressed vector produced greaterthan 40 mg/liter of greater than 90% full-length fusion protein.

Constructs to precisely express the toxin B repeats in either pMa1c(pMB1850-2360) or pET16b (pPB1850-2360) were constructed as follows. TheDNA interval including the toxin B repeats was PCR amplified asdescribed above utilizing PCR primers P14 (SEQ ID NO:19) and P8 (SEQ IDNO:14). Primer P14 adds a EcoRI site immediately flanking the start ofthe toxin B repeats.

The amplified fragment was cloned into the pT7 Blue T-vector (Novagen)and recombinant clones in which single copies of the PCR fragment wereinserted in either orientation were selected (pT71850-2360) andconfirmed by restriction mapping. The insert was excised from twoappropriately oriented independently isolated pT71850-2360 plasmids,with EcoRI (5′ end of repeats) and PstI (in the flanking polylinker ofthe vector), and cloned into EcoRI/PstI cleaved pMa1c vector. Theresulting construct (pMB1850-2360) was confirmed by restrictionanalysis, and yielded 20 mg/l of soluble fusion protein [greater than90% intact (i.e., nondegraded)] after affinity chromatography.Restriction of this plasmid with HindIII and religation of the vectorresulted in the removal of the 1970-2360 interval. The resultantconstruct (pMB1850-1970) expressed greater than 70 mg/liter of 90% fulllength fusion protein.

The pPB1850-2360 construct was made by cloning a EcoRI (filled withKlenow)-BamHI fragment from a pT71850-2360 vector (opposite orientationto that used in the pMB1850-2360 construction) into NdeI (filled)/BamHIcleaved pET16b vector. Yields of affinity purified soluble fusionprotein were 15 mg/liter, of greater than 90% full length fusionprotein.

Several smaller expression constructs from the 1750-2070 interval werealso constructed in His-tagged pET vectors, but expression of theseplasmids in the BL21 (DE3) host resulted in the production of highlevels of mostly insoluble protein (see Table 23 and FIG. 19). Theseconstructs were made as follows. pPB1850-1970 was constructed by cloninga BglII-HindIII fragment of pPB1850-2360 into BglII/HindIII cleavedpET23b vector. pPB1850-2070 was constructed by cloning a BglII-PvuIIfragment of pPB1850-2360 into BglII/HincII cleaved pET23b vector.pPB1750-1970(c) was constructed by removal of the internal HindIIIfragment of a pPB1750-2360 vector in which the vector HindIII site wasregenerated during cloning (presumably by the addition of an A residueto the amplified PCR product by terminal transferase activity of Pfupolymerase). The pPB1750-1970(n) construct was made by insertion of theinsert containing the NdeI-HindIII fragment of pPB1750-2360 intoidentically cleaved pETHisb vector. All constructs were confirmed byrestriction digestion.

An expression construct that directs expression of the 10-470 aainterval of toxin B was constructed in the pMa1c vector as follows. ANheI (a site 5′ to the insert in the pET23 vector)-AfII (filled)fragment of the toxin B gene from pPB 10-1530 was cloned into XbaI(compatible with NheI)/HindIII (filled) pMa1c vector. The integrity ofthe construct (pMB10-470) was verified by restriction mapping and DNAsequencing of the 5′ clone junction using a MBP specific DNA primer (NewEngland Biolabs). However, all expressed protein was degraded to the MBPmonomer MW.

A second construct spanning this interval (aa 10-470) was constructed bycloning the PCR amplification product from a reaction containing the P9(SEQ ID NO:15) and P10 (SEQ ID NO:16) primers (FIG. 18) into the pETHisavector. This was accomplished by cloning the PCR product as anEcoRI-blunt fragment into EcoRI-HincII restricted vector DNA;recombinant clones were verified by restriction mapping. Although thisconstruct (pPB10-520) allowed expression and purification (utilizing theN-terminal polyhistidine affinity tag) of intact fusion protein, yieldswere estimated at less than 500 μg per liter culture.

Higher yield of recombinant protein from this interval (aa 10-520) wereobtained by expression of the interval in two overlapping clones. The10-330aa interval was cloned in both pETHisa and pMa1c vectors, usingthe BamHI-AfIII (filled) DNA fragment from pPB10-520. This fragment wascloned into BamHI-HindIII (filled) restricted pMa1c or BamHI-HincIIrestricted pETHisa vector. Recombinant clones were verified byrestriction mapping. High level expression of either insoluble (pET) orsoluble (pMa1) fusion protein was obtained. Total yields of fusionprotein from the pMB10-330 construct (FIG. 18) were 20 mg/liter culture,of which 10% was estimated to be full-length fusion protein. Althoughyields of this interval were higher and >90% full-length recombinantprotein produced when expressed from the pET construct, the pMa1 fusionwas utilized since the expressed protein was soluble and thus morelikely to have the native conformation.

The pMB260-520 clone was constructed by cloning EcoRI-XbaI cleavedamplified DNA from a PCR reaction containing the P11 (SEQ ID NO:17) andP10 (SEQ ID NO:16) DNA primers (FIG. 18) into similarly restricted pMa1cvector. Yields of affinity purified protein were 10 mg/liter, of whichapproximately 50% was estimated to be full-length recombinant protein.

The aa510-1110 interval was expressed as described below. This entireinterval was expressed as a pMa1 fusion by cloning the NheI-HindIIIfragment of pUCB10-1530 into XbaI-HindIII cleaved pMa1c vector. Theintegrity of the construct (pMB510-1110) was verified by restrictionmapping and DNA sequencing of the 5′ clone junction using a MBP specificDNA primer. The yield of affinity purified protein was 25 mg/literculture, of which 5% was estimated to be full-length fusion protein (1mg/liter).

To attempt to obtain higher yields, this region was expressed in twofragments (aa510-820, and 820-1110) in the pMa1c vector. The pMB510-820clone was constructed by insertion of a SacI (in the pMa1c polylinker 5′to the insert)-HpaI DNA fragment from pMB510-1110 into SacI/StuIrestricted pMa1c vector. The pMB820-1110 vector was constructed byinsertion of the HpaI-HindIII fragment of pUCB10-1530 into BamHI(filled)/HindIII cleaved pMa1c vector. The integrity of these constructswere verified by restriction mapping and DNA sequencing of the 5′ clonejunction using a MBP specific DNA primer.

Recombinant protein expressed from the pMB510-820 vector was highlyunstable. However, high levels (20 mg/liter) of >90% full-length fusionprotein were obtained from the pMB820-1105 construct. The combination ofpartially degraded pMB510-1110 protein (enriched for the 510-820interval) with the pMB820-1110 protein provides usable amounts ofrecombinant antigen from this interval.

The aa1100-1750 interval was expressed as described below. The entireinterval was expressed in the pMa1c vector from a construct in which theAccI(filled)-SpeI fragment of pPB10-1750 was inserted into StuI/XbaI(XbaI is compatible with SpeI; StuI and filled AccI sites are both bluntended) restricted pMa1c. The integrity of this construct (pMB1100-1750)was verified by restriction mapping and DNA sequencing of the clonejunction using a MBP specific DNA primer. Although 15 mg/liter ofaffinity purified protein was isolated from cells harboring thisconstruct, the protein was greater than 99% degraded to MBP monomersize.

A smaller derivative of pMB1100-1750 was constructed by restriction ofpMB1100-1750 with AfII and SalI (in the pMa1c polylinker 3′ to theinsert), filling in the overhanging ends, and religating the plasmid.The resultant clone (verified by restriction digestion and DNAsequencing) has deleted the aa1530-1750 interval, was designatedpMB1100-1530. pMB1100-1530 expressed recombinant protein at a yield ofgreater than 40 mg/liter, of which 5% was estimated to be full-lengthfusion protein.

Three constructs were made to express the remaining interval. Initially,a EspHI (filled)-SpeI fragment from pPB10-1750 was cloned intoEcoRI(filled)/XbaI cleaved pMa1c vector. The integrity of this construct(pMB1570-1750) was verified by restriction mapping and DNA sequencing ofthe 5′ clone junction using a MBP specific DNA primer. Expression ofrecombinant protein from this plasmid was very low, approximately 3 mgaffinity purified protein per liter, and most was degraded to MBPmonomer size. This region was subsequently expressed from a PCRamplified DNA fragment. A PCR reaction utilizing primers P13 [SEQ IDNO:18; P13 was engineered to introduce an EcoRI site 5′ to amplifiedtoxin B sequences] and P8 (SEQ ID NO: 14) was performed on C. difficilegenomic DNA as described above. The amplified fragment was cleaved withEcoRI and SpeI, and cloned into EcoRI/XbaI cleaved pMa1c vector. Theresultant clone (pMB1530-1750) was verified by restriction map analysis,and recombinant protein was expressed and purified. The yield wasgreater than 20 mg protein per liter culture and it was estimated that25% was full-length fusion protein; this was a significantly higheryield than the original pMB1570-1750 clone: The insert of pMB1530-1750(in a EcoRI-SalI fragment) was transferred to the pETHisa vector(EcoRI/XhoI cleaved, XhoI and SalI ends are compatible). No detectablefusion protein was purified on Ni-Chelate columns from soluble lysatesof cells induced to express fusion protein from this construct. TABLE 23Summary Of Toxin B Expression Constructs^(a) Yield Clone Affinity Tag(mg/liter) % Full Length pPB10-1750 none low (estimated ? from Westernblot hyb) pPB10-1530 none low (as above) ? pMB10-470 MBP   15 mg/l    0%pPB10-520 poly-his  0.5 mg/l   20% pPB10-330 poly-his >20 mg/l   90%(insoluble) pMB10-330 MBP   20 mg/l   10% pMB260-520 MBP   10 mg/l   50%pMB510-1110 MBP   25 mg/l    5% pMB510-820 MBP degraded (by Western blothyb) pMB820-1110 MBP   20 mg/l   90% pMB1100-1750 MBP   15 mg/l    0%pMB1100-1530 MBP   40 mg/l    5% pMB1570-1750 MBP    3 mg/l  <5%pPB1530-1750 poly-his no purified ? protein detected pMB1530-1750 MBP  20 mg/l   25% pMB1750-2360 MBP >20 mg/l >90% pMBp1750-2360 MBP  6.5mg/l 50% (secreted) pPB1750-2360 poly-his >20 mg/l >90% pMB1750-1970MBP >20 mg/l >90% pMB1970-2360 MBP   40 mg/l >90% pMBp1970-2360 MBP (nosecretion) NA pMB1850-2360 MBP   20 mg/l >90% pPB1850-2360 poly-his   15mg/l >90% pMB1850-1970 MBP   70 mg/l >90% pPB1850-1970 poly-his >10mg/l >90% (insoluble) pPB1850-2070 poly-his >10 mg/l >90% (insoluble)pPB1750-1970(c) poly-his >10 mg/l >90% (insoluble) pPB1750-1970(n)poly-his >10 mg/l >90% (insoluble)^(a)Clones in italics are clones currently utilized to purifyrecombinant protein from each selected interval.

Example 19 Identification, Purification and Induction of NeutralizingAntibodies Against Recombinant C. difficile Toxin B Protein

To determine whether recombinant toxin B polypeptide fragments cangenerate neutralizing antibodies, typically animals would first beimmunized with recombinant proteins and anti-recombinant antibodies aregenerated. These anti-recombinant protein antibodies are then tested forneutralizing ability in vivo or in vitro. Depending on the immunogenicnature of the recombinant polypeptide, the generation of high-titerantibodies against that protein may take several months. To acceleratethis process and identify which recombinant polypeptide(s) may be thebest candidate to generate neutralizing antibodies, depletion studieswere performed. Specifically, recombinant toxin B polypeptide werepre-screened by testing whether they have the ability to bind toprotective antibodies from a CTB antibody preparation and hence depletethose antibodies of their neutralizing capacity. Those recombinantpolypeptides found to bind CTB, were then utilized to generateneutralizing antibodies. This Example involved: a) identification ofrecombinant sub-regions within toxin B to which neutralizing antibodiesbind; b) identification of toxin B sub-region specific antibodies thatneutralize toxin B in vivo; and c) generation and evaluation ofantibodies reactive to recombinant toxin B polypeptides.

a) Identification of Recombinant Sub-Regions Within Toxin B to WhichNeutralizing Antibodies Bind

Sub-regions within toxin B to which neutralizing antibodies bind wereidentified by utilizing recombinant toxin B proteins to depleteprotective antibodies from a polyclonal pool of antibodies againstnative C. difficile toxin B. An in vivo assay was developed to evaluateprotein preparations for the ability to bind neutralizing antibodies.Recombinant proteins were first pre-mixed with antibodies directedagainst native toxin B (CTB antibody; see Example 8) and allowed toreact for one hour at 37° C. Subsequently, C. difficile toxin B (CTB;Tech Lab) was added at a concentration lethal to hamsters and incubatedfor another hour at 37° C. After incubation this mixture was injectedintraperitoneally (IP) into hamsters. If the recombinant polypeptidecontains neutralizing epitopes, the CTB antibodies will lose its abilityto protect the hamsters against death from CTB. If partial or completeprotection occurs with the CTB antibody-recombinant mixture, thatrecombinant contains only weak or non-neutralizing epitopes of toxin B.This assay was performed as follows.

Antibodies against CTB were generated in egg laying Leghorn hens asdescribed in Example 8. The lethal dosage (LD₁₀₀) of C. difficile toxinB when delivered I.P. into 40 g female Golden Syrian hamsters (CharlesRiver) was determined to be 2.5 to 5 μg. Antibodies generated againstCTB and purified by PEG precipitation could completely protect thehamsters at the I.P. dosage determined above. The minimal amount of CTBantibody needed to afford good protection against 5 μg of CTB wheninjected I.P. into hamsters was also determined (1×PEG prep). Theseexperiments defined the parameters needed to test whether a givenrecombinant protein could deplete protective CTB antibodies.

The cloned regions tested for neutralizing ability cover the entiretoxin B gene and were designated as Intervals (INT) 1 through 5 (seeFIG. 19). Approximately equivalent final concentrations of eachrecombinant polypeptide were tested. The following recombinantpolypeptides were used: 1) a mixture of intervals 1 and 2 (INT-1,2); 2)a mixture of Intervals 4 and 5 (INT-4, 5) and 3) Interval 3 (INT-3).Recombinant proteins (each at about 1 mg total protein) were firstpreincubated with a final CTB antibody concentration of 1× [i.e., pelletdissolved in original yolk volume as described in Example 1(c)] in afinal volume of 5 mls for 1 hour at 37° C. Twenty-five μg of CTB (at aconcentration of 5 μg/ml), enough CTB to kill 5 hamsters, was then addedand the mixture was then incubated for 1 hour at 37° C. Five, 40 gfemale hamsters (Charles River) in each treatment group were then eachgiven 1 ml of the mixture I.P. using a tuberculin syringe with a 27gauge needle. The results of this experiment are shown in Table 24.TABLE 24 Binding Of Neutralizing Antibodies By INT 3 Protein Number OfAnimals Number Of Animals Treatment Group¹ Alive Dead CTB antibodies 3 2CTB antibodies + INT1, 2 3 2 CTB antibodies + INT4, 5 3 2 CTBantibodies + INT 3 0 5¹ C. difficile toxin B (CTB) was added to each group.¹ C. difficile toxin B (CTB) was added to each group.

As shown in Table 24, the addition of recombinant proteins from INT-1, 2or INT-4, 5 had no effect on the in vivo protective ability of the CTBantibody preparation compared to the CTB antibody preparation alone. Incontrast, INT-3 recombinant polypeptide was able to remove all of thetoxin B neutralizing ability of the CTB antibodies as demonstrated bythe death of all the hamsters in that group.

The above experiment was repeated, using two smaller expressed fragments(pMB 1750-1970 and pMB 1970-2360, see FIG. 19) comprising the INT-3domain to determine if that domain could be further subdivided intosmaller neutralizing epitopes. In addition, full-length INT-3polypeptide expressed as a nickel tagged protein (pPB1750-2360) wastested for neutralizing ability and compared to the original INT-3expressed MBP fusion (pMB1750-2360). The results are shown in Table 25.TABLE 25 Removal Of Neutralizing Antibodies By Repeat ContainingProteins Number Of Number Of Animals Animals Treatment Group¹ Alive DeadCTB antibodies 5 0 CTB antibodies + pPB1750-2360 0 5 CTB antibodies +pMB1750-2360 0 5 CTB antibodies + pMB1970-2360 3 2 CTB antibodies +pMB1750-1970 2 3¹ C. difficile toxin B (CTB) was added to each group.

The results summarized in Table 25 indicate that the smaller polypeptidefragments within the INT-3 domain, pMB1750-1970 and pMB1970-2360,partially lose the ability to bind to and remove neutralizing antibodiesfrom the CTB antibody pool. These results demonstrate that the fulllength INT-3 polypeptide is required to completely deplete the CTBantibody pool of neutralizing antibodies. This experiment also showsthat the neutralization epitope of INT-3 can be expressed in alternativevector systems and the results are independent of the vector utilized orthe accompanying fusion partner.

Other Interval 3 specific proteins were subsequently tested for theability to remove neutralizing antibodies within the CTB antibody poolas described above. The Interval 3 specific proteins used in thesestudies are summarized in FIG. 23. In FIG. 23 the followingabbreviations are used: pP refers to the pET23 vector; pM refers to thepMALc vector; B refers to toxin B; the numbers refer to the amino acidinterval expressed in the clone. The solid black ovals represent theMBP; and HHH represents the poly-histidine tag.

Only recombinant proteins comprising the entire toxin B repeat domain(pMB1750-2360, pPB1750-2360 and pPB1850-2360) can bind and completelyremove neutralizing antibodies from the CTB antibody pool. Recombinantproteins comprising only a portion of the toxin B repeat domain were notcapable of completely removing neutralizing antibodies from the CTBantibody pool (pMB1750-1970 and pMB1970-2360 could partially removeneutralizing antibodies while pMB1850-1970 and pPB1850-2070 failed toremove any neutralizing antibodies from the CTB antibody pool).

The above results demonstrate that only the complete ligand bindingdomain (repeat region) of the toxin B gene can bind and completelyremove neutralizing antibodies from the CTB antibody pool. These resultsdemonstrate that antibodies directed against the entire toxin B repeatregion are necessary for in vivo toxin neutralization (see FIG. 23; onlythe recombinant proteins expressed by the pMB1750-2360, pPB1750-2360 andpPB1850-2360 vectors are capable of completely removing the neutralizingantibodies from the CTB antibody pool).

These results represent the first indication that the entire repeatregion of toxin B would be necessary for the generation of antibodiescapable of neutralizing toxin B, and that sub-regions may not besufficient to generate maximal titers of neutralizing antibodies.

b) Identification of Toxin B Sub-Region Specific Antibodies thatNeutralize Toxin B In Vivo

To determine if antibodies directed against the toxin B repeat regionare sufficient for neutralization, region specific antibodies within theCTB antibody preparation were affinity purified, and tested for in vivoneutralization. Affinity columns containing recombinant toxin B repeatproteins were made as described below. A separate affinity column wasprepared using each of the following recombinant toxin B repeatproteins: pPB750-2360, pPB850-2360, pMB1750-1970 and pMB1970-2360.

For each affinity column to be made, four ml of PBS-washed Actigel resin(Sterogene) was coupled overnight at room temperature with 5-10 mg ofaffinity purified recombinant protein (first extensively dialyzed intoPBS) in 15 ml tubes (Falcon) containing a 1/10 final volume Ald-couplingsolution (1 M sodium cyanoborohydride). Aliquots of the supernatantsfrom the coupling reactions, before and after coupling, were assessed byCoomassie staining of 7.5% SDS-PAGE gels. Based on protein bandintensities, in all cases greater than 30% coupling efficiencies wereestimated. The resins were poured into 10 ml columns (BioRad), washedextensively with PBS, pre-eluted with 4M guanidine-HCl (in 10 mMTris-HCl, pH 8.0) and reequilibrated in PBS. The columns were stored at4° C.

Aliquots of a CTB IgY polyclonal antibody preparation (PEG prep) wereaffinity purified on each of the four columns as described below. Thecolumns were hooked to a UV monitor (ISCO), washed with PBS and 40 mlaliquots of a 2×PEG prep (filter sterilized using a 0.45μ filter) wereapplied. The columns were washed with PBS until the baseline value wasre-established. The columns were then washed with BBStween to elutenonspecifically binding antibodies, and reequilibrated with PBS. Boundantibody was eluted from the column in 4M guanidine-HCl (in 10 nMTris-HCl, pH 8.0). The eluted antibody was immediately dialyzed againsta 100-fold excess of PBS at 4° C. for 2 hrs. The samples were thendialyzed extensively against at least 2 changes of PBS, and affinitypurified antibody was collected and stored at 4° C. The antibodypreparations were quantified by UV absorbance. The elution volumes werein the range of 4-8 ml. All affinity purified stocks contained similartotal antibody concentrations, ranging from 0.25-0.35% of the totalprotein applied to the columns.

The ability of the affinity purified antibody preparations to neutralizetoxin B in vivo was determined using the assay outlined in a) above.Affinity purified antibody was diluted 1:1 in PBS before testing. Theresults are shown in Table 26.

In all cases similar levels of toxin neutralization was observed, suchthat lethality was delayed in all groups relative to preimmune controls.This result demonstrates that antibodies reactive to the repeat regionof the toxin B gene are sufficient to neutralize toxin B in vivo. Thehamsters will eventually die in all groups, but this death is maximallydelayed with the CTB PEG prep antibodies. Thus neutralization with theaffinity purified (AP) antibodies is not as complete as that observedwith the CTB prep before affinity chromatography. This result may be dueto loss of activity during guanidine denaturation (during the elution ofthe antibodies from the affinity column) or the presence of antibodiesspecific to other regions of the toxin B gene that can contribute totoxin neutralization (present in the CTB PEG prep). TABLE 26Neutralization Of Toxin B By Affinity Purified Antibodies TreatmentNumber Animals Number Animals group^(a) Alive^(b) Dead^(b) Preimmune¹ 05 CTB¹; 400 μg 5 0 CTB (AP on pPB1750-2360);² 5 0 875 μg CTB (AP onpMB1750-1970);² 5 0 875 μg CTB (AP on pMB1970-2360);² 5 0 500 μg^(a) C. difficile toxin B (CTB) (Tech Lab; at 5 μg/ml, 25 μg total) atlethal concentration to hamsters is added to antibody and incubated forone hour at 37° C. After incubation this mixture is injectedintraperitoneally (IP) into hamsters. Each treatment group # receivedtoxin premixed with antibody raised against the indicated protein, aseither: ¹ 4X antibody# PEG prep or ²affinity purified (AP) antibody (from CTB PEG prep, onindicated columns). The amount of specific antibody in each prep isindicated; the amount is directly determined for affinity purified prepsand is estimated for the 4X # CTB as described in Example 15.^(b)The numbers in each group represent numbers of hamsters dead oralive, 2 hr post IP administration of toxin/antibody mixture.

The observation that antibodies affinity purified against thenon-overlapping pMB1750-1970 and pMB1970-2360 proteins neutralized toxinB raised the possibility that either 1) antibodies specific to repeatsub-regions are sufficient to neutralize toxin B or 2) sub-regionspecific proteins can bind most or all repeat specific antibodiespresent in the CTB polyclonal pool. This would likely be due toconformational similarities between repeats, since homology in theprimary amino acid sequences between different repeats is in the rangeof only 25-75% [Eichel-Streiber, et al. (1992) Molec. Gen. Genetics233:260]. These possibilities were tested by affinity chromatography.

The CTB PEG prep was sequentially depleted 2× on the pMB1750-1970column; only a small elution peak was observed after the secondchromatography, indicating that most reactive antibodies were removed.This interval depleted CTB preparation was then chromatographed on thepPB 1850-2360 column; no antibody bound to the column. The reactivity ofthe CTB and CTB (pMB1750-1970 depleted) preps to pPB1750-2360,pPB1850-2360, pMB1750-1970 and pMB1970-2360 proteins was then determinedby ELISA using the protocol described in Example 13(c). Briefly, 96-wellmicrotiter plates (Falcon, Pro-Bind Assay Plates) were coated withrecombinant protein by adding 100 μl volumes of protein at 1-2 μg/ml inPBS containing 0.005% thimerosal to each well and incubating overnightat 4° C. The next morning, the coating suspensions were decanted and thewells were washed three times using PBS. In order to block non-specificbinding sites, 100 μl of 1.0% BSA (Sigma) in PBS (blocking solution) wasthen added to each well, and the plates were incubated for 1 hr. at 37°C. The blocking solution was decanted and duplicate samples of 150 μl ofdiluted antibody was added to the first well of a dilution series. Theinitial testing serum dilution was 1/200 for CTB prep, (theconcentration of depleted CTB was standardized by OD₂₈₀) in blockingsolution containing 0.5% Tween 20, followed by 5-fold serial dilutionsinto this solution. This was accomplished by serially transferring 30 μlaliquots to 120 μl buffer, mixing, and repeating the dilution into afresh well. After the final dilution, 30 μl was removed from the wellsuch that all wells contained 120 μl final volume. A total of 5 suchdilutions were performed (4 wells total). The plates were incubated for1 hr at 37° C. Following this incubation, the serially diluted sampleswere decanted and the wells were washed three times using PBS containing0.5% Tween 20 (PBST), followed by two 5 min washes using BBS-Tween and afinal three washes using PBST. To each well, 100 μl of 1/1000 dilutedsecondary antibody [rabbit anti-chicken IgG alkaline phosphatase (Sigma)diluted in blocking solution containing 0.5% Tween 20] was added, andthe plate was incubated 1 hr at 37° C. The conjugate solutions weredecanted and the plates were washed 6 times in PBST, then once in 50 mMNa₂CO₃, 10 mM MgCl₂, pH 9.5. The plates were developed by the additionof 100 μl of a solution containing 1 mg/ml para-nitro phenyl phosphate(Sigma) dissolved in 50 mM Na₂CO₃, 10 mM MgCl₂, pH 9.5 to each well. Theplates were then incubated at room temperature in the dark for 5-45 min.The absorbency of each well was measured at 410 nm using a Dynatech MR700 plate reader.

As predicted by the affinity chromatography results, depletion of theCTB prep on the pMB1750-1970 column removed all detectable reactivity tothe pMB1970-2360 protein. The reciprocal purification of a CTB prep thatwas depleted on the pMB1970-2360 column yielded no bound antibody whenchromatographed on the pMB1750-1970 column. These results demonstratethat all repeat reactive antibodies in the CTB polyclonal pool recognizea conserved structure that is present in non-overlapping repeats.Although it is possible that this conserved structure represents rareconserved linear epitopes, it appears more likely that the neutralizingantibodies recognize a specific protein conformation. This conclusionwas also suggested by the results of Western blot hybridization analysisof CTB reactivity to these recombinant proteins.

Western blots of 7.5% SDS-PAGE gels, loaded and electrophoresed withdefined quantities of each recombinant protein, were probed with the CTBpolyclonal antibody preparation. The blots were prepared and developedwith alkaline phosphatase as described in Example 3. The results areshown in FIG. 24.

FIG. 24 depicts a comparison of immunoreactivity of IgY antibody raisedagainst either native or recombinant toxin B antigen. Equal amounts ofpMB1750-1970 (lane 1), pMB1970-2360 (lane 2), pPB1850-2360 (lane 3) aswell as a serial dilution of pPB1750-2360 (lanes 4-6 comprising 1×,1/10× and 1/100 amounts, respectively) proteins were loaded in duplicateand resolved on a 7.5% SDS-PAGE gel. The gel was blotted and each halfwas hybridized with PEG prep IgY antibodies from chickens immunized witheither native CTB or pPB1750-2360 protein. Note that the full-lengthpMB1750-1970 protein was identified only by antibodies reactive to therecombinant protein (arrows).

Although the CTB prep reacts with the pPB1750-2360, pPB1850-2360, andpMB1970-2360 proteins, no reactivity to the pMB1750-1970 protein wasobserved (FIG. 24). Given that all repeat reactive antibodies can bebound by this protein during affinity chromatography, this resultindicates that the protein cannot fold properly on Western blots. Sincethis eliminates all antibody reactivity, it is unlikely that the repeatreactive antibodies in the CTB prep recognize linear epitopes. This mayindicate that in order to induce protective antibodies, recombinanttoxin B protein will need to be properly folded.

c) Generation and Evaluation of Antibodies Reactive to Recombinant ToxinB Polypeptides

i) Generation of Antibodies Reactive to Recombinant Toxin B Proteins

Antibodies against recombinant proteins were generated in egg layingLeghorn hens as described in Example 13. Antibodies were raised [usingFreunds adjuvant (Gibco) unless otherwise indicated] against thefollowing recombinant proteins: 1) a mixture of Interval 1+2 proteins(see FIG. 18); 2) a mixture of interval 4 and 5 proteins (see FIG. 18);3) pMB1970-2360 protein; 4) pPB1750-2360 protein; 5) pMB1750-2360; 6)pMB1750-2360 [Titermax adjuvant (Vaxcell)]; 7) pMB1750-2360 [Gerbuadjuvant (Biotech)]; 8) pMBp1750-2360 protein; 9) pPB1850-2360; and 10)pMB1850-2360.

Chickens were boosted at least 3 times with recombinant protein untilELISA reactivity [using the protocol described in b) above with theexception that the plates were coated with pPB1750-2360 protein] ofpolyclonal PEG preps was at least equal to that of the CTB polyclonalantibody PEG prep. ELISA titers were determined for the PEG preps fromall of the above immunogens and were found to be comparable ranging from1:12500 to 1:62500. High titers were achieved in all cases except in 6)pMB 1750-2360 in which strong titers were not observed using theTitermax adjuvant, and this preparation was not tested further.

ii) Evaluation of Antibodies Reactive to Recombinant Proteins by WesternBlot Hybridization

Western blots of 7.5% SDS-PAGE gels, loaded and electrophoresed withdefined quantities of recombinant protein (pMB1750-1970, pPB1850-2360,and pMB1970-2360 proteins and a serial dilution of the pPB1750-2360 toallow quantification of reactivity), were probed with the CTB,pPB1750-2360, pMB1750-2360 and pMB1970-2360 polyclonal antibodypreparations (from chickens immunized using Freunds adjuvant). The blotswere prepared and developed with alkaline phosphatase as described abovein b).

As shown in FIG. 24, the CTB and pMB1970-2360 preps reacted stronglywith the pPB1750-2360, pPB1850-2360, and pMB1970-2360 proteins while thepPB1750-2360 and pMB1970-2360 (Gerbu) preparations reacted strongly withall four proteins. The Western blot reactivity of the pPB1750-2360 andpMB1970-2360 (Gerbu) preparations were equivalent to that of the CTBpreparation, while reactivity of the pMB1970-2360 preparation was <10%that of the CTB prep. Despite equivalent ELISA reactivities only weakreactivity (approximately 1%) to the recombinant proteins were observedin PEG preps from two independent groups immunized with the pMB1750-2360protein and one group immunized with the pMB1750-2360 preparation usingFreunds adjuvant.

Affinity purification was utilized to determine if this difference inimmunoreactivity by Western blot analysis reflects differing antibodytiters. Fifty ml 2×PEG preparations from chickens immunized with eitherpMB 1750-2360 or pMB1970-2360 protein were chromatographed on thepPB1750-2360 affinity column from b) above, as described. The yield ofaffinity purified antibody (% total protein in preparation) wasequivalent to the yield obtained from a CTB PEG preparation in b) above.Thus, differences in Western reactivity reflect a qualitative differencein the antibody pools, rather than quantitative differences. Theseresults demonstrate that certain recombinant proteins are more effectiveat generating high affinity antibodies (as assayed by Western blothybridization).

iii) In Vivo Neutralization of Toxin B Using Antibodies Reactive toRecombinant Protein

The in vivo hamster model [described in Examples 9 and 14(b)] wasutilized to assess the neutralizing ability of antibodies raised againstrecombinant toxin B proteins. The results from three experiments areshown below in Tables 27-29.

The ability of each immunogen to neutralize toxin B in vivo has beencompiled and is shown in Table 30. As predicted from the recombinantprotein-CTB premix studies (Table 24) only antibodies to Interval 3(1750-2366) and not the other regions of toxin B (i.e., intervals 1-5)are protective. Unexpectedly, antibodies generated to INT-3 regionexpressed in pMAL vector (pMB1750-2360 and pMB1750-2360) using Freundsadjuvant were non-neutralizing. This observation is reproducible, sinceno neutralization was observed in two independent immunizations withpMB1750-2360 and one immunization with pMB1750-2360. The fact that 5×quantities of affinity purified toxin B repeat specific antibodies frompMB1750-2360 PEG preps cannot neutralize toxin B while 1× quantities ofaffinity purified anti-CTB antibodies can (Table 28) demonstrates thatthe differential ability of CTB antibodies to neutralize toxin B is dueto qualitative rather than quantitative differences in these antibodypreparations. Only when this region was expressed in an alternativevector (pPB1750-2360) or using an alternative adjuvant with thepMB1750-2360 protein were neutralizing antibodies generated.Importantly, antibodies raised using Freunds adjuvant to pPB1850-2360,which contains a fragment that is only 100 amino acids smaller thanrecombinant pPB1750-2360, are unable to neutralize toxin B in vivo(Table 27); note also that the same vector is used for both pPB1850-2360and pPB1750-2360. TABLE 27 In vivo Neutralization Of Toxin B TreatmentNumber Animals Number Animals Group^(a) Alive^(b) Dead^(b) Preimmune 0 5CTB 5 0 INT1 + 2 0 5 INT 4 + 5 0 5 pMB1750-2360 0 5 pMB1970-2360 0 5pPB1750-2360 5 0^(a) C. difficile toxin B (CTB) (at 5 μg/ml; 25 μg total; Tech Lab) atlethal concentration to hamsters is added to antibody and incubated forone hour at 37° C. After incubation this mixture is injectedintraperitoneally (IP) into hamsters. Each treatment group receivedtoxin premixed with antibody raised against the indicated protein, as a4X antibody PEG prep.^(b)The numbers in each group represent numbers of hamsters dead oralive, 2 hours post IP administration of toxin/antibody mixture.

TABLE 28 In vivo Neutralization Of Toxin B Using Affinity PurifiedAntibodies Treatment Number Animals Number Animals Group^(a) Alive^(b)Dead^(b) Preimmune(1) 0 5 CTB(1) 5 0 pPB1750-2360(1) 5 0 1.5 mganti-pMB1750-2360(2) 1 4 1.5 mg anti-pMB1970-2360(2) 0 5 300 μganti-CTB(2) 5 0^(a) C. difficile toxin B (CTB) (at 5 μg/ml; 25 μg total; Tech Lab) atlethal concentration to hamsters is added to antibody and incubated forone hour at 37° C. After incubation, 1 ml of this mixture is injectedintraperitoneally (IP) into hamsters. Each treatment group receivedtoxin premixed with antibody raised against the indicated protein, aseither (1) 4X antibody PEG# prep or (2) affinity purified antibody (on a pPB1750-2360 resin),either 1.5 mg/group (anti-pMB1750-2360 and anti-pMB1970-2360; usedundiluted affinity purified antibody) or 350 μg/group (anti-CTB, repeatspecific; used 1/5 diluted anti-CTB antibody).^(b)The numbers in each group represent numbers of hamsters dead oralive, 2 hr post-IP administration of toxin/antibody mixture.

TABLE 29 Generation Of Neutralizing Antibodies Utilizing The GerbuAdjuvant Number Animals Number Animals Treatment Group^(a) Alive^(b)Dead^(b) Preimmune 0 5 CTB 5 0 pMB1970-2360 0 5 pMB1850-2360 0 5pPB1850-2360 0 5 pMB1750-2360 5 0 (Gerbu adj)^(a) C. difficile toxin B (CTB) (Tech Lab) at lethal concentration tohamsters is added to antibody and incubated for one hour at 37° C. Afterincubation this mixture is injected intraperitoneally (IP) intohamsters. Each treatment group received toxin premixed with antibodyraised against the indicated protein, as a 4X antibody PEG prep.^(b)The numbers in each group represent numbers of hamsters dead oralive, 2 hrs post IP administration of toxin/antibody mixture.

TABLE 30 In vivo Neutralization Of Toxin B Antigen In vivo TestedUtilized Neu- Immunogen Adjuvant Preparation^(a) For AP tralization^(b)Preimmune NA¹ PEG NA no CTB Titermax PEG NA yes (native) CTB Titermax APpPB1750-2360 yes (native) CTB Titermax AP pPB1850-2360 yes (native) CTBTitermax AP pPB1750-1970 yes (native) CTB Titermax AP pPB1970-2360 yes(native) pMB1750-2360 Freunds PEG NA no pMB1750-2360 Freunds APpPB1750-2360 no pMB1750-2360 Gerbu PEG NA yes pMB1970-2360 Freunds PEGNA no pMB1970-2360 Freunds AP pPB1750-2360 no pPB1750-2360 Freunds PEGNA yes pPB1850-2360 Freunds PEG NA no pMB1850-2360 Freunds PEG NA no INT1 + 2 Freunds PEG NA no INT 4 + 5 Freunds PEG NA no^(a)Either PEG preparation (PEG) or affinity purified antibodies (AP).^(b)‘Yes’ denotes complete neutralization (0/5 dead) while ‘no’ denotesno neutralization (5/5 dead) of toxin B, 2 hours post-administration ofmixture.¹‘NA’ denotes not applicable.

The pPB1750-2360 antibody pool confers significant in vivo protection,equivalent to that obtained with the affinity purified CTB antibodies.This correlates with the observed high affinity of this antibody pool(relative to the pMB 1750-2360 or pMB1970-2360 pools) as assayed byWestern blot analysis (FIG. 24). These results provide the firstdemonstration that in vivo neutralizing antibodies can be induced usingrecombinant toxin B protein as immunogen.

The failure of high concentrations of antibodies raised against thepMB1750-2360 protein (using Freunds adjuvant) to neutralize, while theuse of Gerbu adjuvant and pMB1750-2360 protein generates a neutralizingresponse, demonstrates that conformation or presentation of this proteinis essential for the induction of neutralizing antibodies. These resultsare consistent with the observation that the neutralizing antibodiesproduced when native CTB is used as an immunogen appear to recognizeconformational epitopes [see section b) above]. This is the firstdemonstration that the conformation or presentation of recombinant toxinB protein is essential to generate high titers of neutralizingantibodies.

Example 20 Determination of Quantitative and Qualitative DifferencesBetween pMB1750-2360, pMB1750-2360 (Gerbu) or pPB1750-2360 IgYPolyclonal Antibody Preparations

In Example 19, it was demonstrated that toxin B neutralizing antibodiescould be generated using specific recombinant toxin B proteins(pPB1750-2360) or specific adjuvants. Antibodies raised againstpMB1750-2360 were capable of neutralizing the enterotoxin effect oftoxin B when the recombinant protein was used to immunize hens inconjunction with the Gerbu adjuvant, but not when Freunds adjuvant wasused. To determine the basis for these antigen and adjuvantrestrictions, toxin B-specific antibodies present in the neutralizingand non-neutralizing PEG preparations were isolated by affinitychromatography and tested for qualitative or quantitative differences.The example involved a) purification of anti-toxin B specific antibodiesfrom pMB1750-2360 and pPB1750-2360 PEG preparations and b) in vivoneutralization of toxin B using the affinity purified antibody.

a) Purification of Specific Antibodies from pMB1750-2360 andpPB1750-2360 PEG Preparations

To purify and determine the concentration of specific antibodies(expressed as the percent of total antibody) within the pPB1750-2360(Freunds and Gerbu) and pPB1750-2360 PEG preparations, definedquantities of these antibody preparations were chromatographed on anaffinity column containing the entire toxin B repeat region (pPB1750-2360). The amount of affinity purified antibody was thenquantified.

An affinity column containing the recombinant toxin B repeat protein,pPB1750-2360, was made as follows. Four ml of PBS-washed Actigel resin(Sterogene) was coupled with 5 mg of pPB1750-2360 affinity purifiedprotein (dialyzed into PBS; estimated to be greater than 95% full lengthfusion protein) in a 15 ml tube (Falcon) containing 1/10 final volumeAid-coupling solution (1M sodium cyanoborohydride). Aliquots of thesupernatant from the coupling reactions, before and after coupling, wereassessed by Coomassie staining of 7.5% SDS-PAGE gels. Based on proteinband intensities, greater than 95% (approximately 5 mg) of recombinantprotein was coupled to the resin. The coupled resin was poured into a 10ml column (BioRad), washed extensively with PBS, pre-eluted with 4Mguanidine-HCl (in 10 mM Tris-HCl, pH 8.0; 0.005% thimerosal) andre-equilibrated in PBS and stored at 4° C.

Aliquots of pMB1750-2360, pMB1750-2360 (Gerbu) or pPB1750-2360 IgYpolyclonal antibody preparations (PEG preps) were affinity purified onthe above column as follows. The column was attached to an UV monitor(ISCO), and washed with PBS. Forty ml aliquots of 2×PEG preps (filtersterilized using a 0.45μ filter and quantified by OD₂₈₀ beforechromatography) was applied. The column was washed with PBS until thebaseline was re-established (the column flow-through was saved), washedwith BBSTween to elute nonspecifically binding antibodies andre-equilibrated with PBS. Bound antibody was eluted from the column in4M guanidine-HCl (in 10 mM Tris-HCL, pH 8.0, 0.005% thimerosal) and theentire elution peak collected in a 15 ml tube (Falcon). The column wasre-equilibrated, and the column eluate re-chromatographed as describedabove. The antibody preparations were quantified by UV absorbance (theelution buffer was used to zero the spectrophotometer). Approximately 10fold higher concentrations of total purified antibody was obtained uponelution of the first chromatography pass relative to the second pass.The low yield from the second chromatography pass indicated that most ofthe specific antibodies were removed by the first round ofchromatography.

Pools of affinity purified specific antibodies were prepared by dialysisof the column elutes after the first column chromatography pass for thepMB1750-2360, pMB1750-2360 (Gerbu) or pPB1750-2360 IgY polyclonalantibody preparations. The elutes were collected on ice and immediatelydialyzed against a 100-fold volume of PBS at 4° C. for 2 hrs. Thesamples were then dialyzed against 3 changes of a 65-fold volume of PBSat 4° C. Dialysis was performed for a minimum of 8 hrs per change ofPBS. The dialyzed samples were collected, centrifuged to removeinsoluble debris, quantified by OD₂₈₀, and stored at 4° C.

The percentage of toxin B repeat-specific antibodies present in eachpreparation was determined using the quantifications of antibody yieldsfrom the first column pass (amount of specific antibody recovered afterfirst pass/total protein loaded). The yield of repeat-specific affinitypurified antibody (expressed as the percent of total protein in thepreparation) in: 1) the pMB1750-2360 PEG prep was approximately 0.5%, 2)the pMB1750-2360 (Gerbu) prep was approximately 2.3%, and 3) thepPB1750-2360 prep was approximately 0.4%. Purification of a CTB IgYpolyclonal antibody preparation on the same column demonstrated that theconcentration of toxin B repeat specific antibodies in the CTBpreparation was 0.35%.

These results demonstrate that 1) the use of Gerbu adjuvant enhanced thetiter of specific antibody produced against the pMB1750-2360 protein5-fold relative to immunization using Freunds adjuvant, and 2) thedifferences seen in the in vivo neutralization ability of thepMB1750-2360 (not neutralizing) and pPB1750-2360 (neutralizing) and CTB(neutralizing) PEG preps seen in Example 19 was not due to differencesin the titers of repeat-specific antibodies in the three preparationsbecause the titer of repeat-specific antibody was similar for all threepreps; therefore the differing ability of the three antibodypreparations to neutralize toxin B must reflect qualitative differencesin the induced toxin B repeat-specific antibodies. To confirm thatqualitative differences exist between antibodies raised in hensimmunized with different recombinant proteins and/or differentadjuvants, the same amount of affinity purified anti-toxin B repeat (aa1870-2360 of toxin B) antibodies from the different preparations wasadministered to hamsters using the in vivo hamster model as describedbelow.

b) In Vivo Neutralization of Toxin B Using Affinity Purified Antibody

The in vivo hamster model was utilized to assess the neutralizingability of the affinity purified antibodies raised against recombinanttoxin B proteins purified in (a) above. As well, a 4×IgY PEG preparationfrom a second independent immunization utilizing the pPB1750-2360antigen with Freunds adjuvant was tested for in vivo neutralization. Theresults are shown in Table 31.

The results shown in Table 31 demonstrate that:

-   1) as shown in Example 19 and reproduced here, 1.5 mg of affinity    purified antibody from pMB1750-2360 immunized hens using Freunds    adjuvant does not neutralize toxin B in vivo. However, 300 μg of    affinity purified antibody from similarly immunized hens utilizing    Gerbu adjuvant demonstrated complete neutralization of toxin B in    vivo. This demonstrates that Gerbu adjuvant, in addition to    enhancing the titer of antibodies reactive to the pMB1750-2360    antigen relative to Freunds adjuvant (demonstrated in (a) above),    also enhances the yield of neutralizing antibodies to this antigen,    greater than 5 fold.-   2) Complete in vivo neutralization of toxin B was observed with 1.5    mg of affinity purified antibody from hens immunized with    pPB1750-2360 antigen, but not with pMB1750-2360 antigen, when    Freunds adjuvant was used. This demonstrates, using standardized    toxin B repeat-specific antibody concentrations, that neutralizing    antibodies were induced when pPB1750-2360 but not pMB1750-2360 was    used as the antigen with Freunds adjuvant.-   3) Complete in vivo neutralization was observed with 300 μg of    pMB1750-2360 (Gerbu) antibody, but not with 300 μg of pPB1750-2360    (Freunds) antibody. Thus the pMB1750-2360 (Gerbu) antibody has a    higher titer of neutralizing antibodies than the pPB1750-2360    (Freunds) antibody.-   4) Complete neutralization of toxin B was observed using 300 μg of    CTB antibody [affinity purified (AP)] but not 100 μg CTB antibody    (AP or PEG prep). This demonstrates that greater than 100 μg of    toxin B repeat-specific antibody (anti-CTB) is necessary to    neutralize 25 μg toxin B in vivo in this assay, and that affinity    purified antibodies specific to the toxin B repeat interval    neutralize toxin B as effectively as the PEP prep of IgY raised    against the entire CTB protein (shown in this assay).

5) As was observed with the initial pPB1750-2360 (IgY) PEG preparation(Example 19), complete neutralization was observed with a IgY PEGpreparation isolated from a second independent group of pPB1750-2360(Freunds) immunized hens. This demonstrates that neutralizing antibodiesare reproducibly produced when hens are immunized with pPB1750-2360protein utilizing Freunds adjuvant. TABLE 31 In vivo Neutralization OfToxin B Using Affinity Purified Antibodies Number Animals Number AnimalsTreatment Group^(a) Alive^(b) Dead^(b) Preimmune¹ 0 5 CTB (300 μg)² 5 0CTB (100 μg)² 1 4 pMB1750-2360 (G) 5 0 (5 mg)² pMB1750-2360 (G) 5 0 (1.5mg)² pMB1750-2360 (G) 5 0 (300 μg)² pMB1750-2360 (F) 0 5 (1.5 mg)²pPB1750-2360 (F) 5 0 (1.5 mg)² pPB1750-2360 (F) 1 4 (300 μg)² CTB (100μg)³ 2 3 pPB1750-2360 (F) 5 0 (500 μg)¹^(a) C. difficile toxin B (CTB) (Tech Lab) at lethal concentration tohamsters (25 μg) was added to the antibody (amount of specific antibodyis indicated) and incubated for one hour at 37° C. After incubation,this mixture was injected IP into hamsters (⅕ total # mix injected perhamster). Each treatment group received toxin premixed with antibodyraised against the indicated protein (G = gerbu adjuvant, F = Freundsadjuvant).¹indicates the antibody was a 4X IgY PEG prep;²indicates the antibody was affinity purified on a pPB1850-2360 resin;and³indicates that the antibody was a 1X IgY PEG prep.^(b)The numbers in each group represent numbers of hamsters dead oralive, 2 hrs post IP administration of toxin/antibody mixture.

Example 21 Diagnostic Enzyme Immunoassays for C. difficile Toxins A andB

The ability of the recombinant toxin proteins and antibodies raisedagainst these recombinant proteins (described in the above examples) toform the basis of diagnostic assays for the detection of clostridialtoxin in a sample was examined. Two immunoassay formats were tested toquantitatively detect C. difficile toxin A and toxin B from a biologicalspecimen. The first format involved a competitive assay in which a fixedamount of recombinant toxin A or B was immobilized on a solid support(e.g., microtiter plate wells) followed by the addition of atoxin-containing biological specimen mixed with affinity-purified or PEGfractionated antibodies against recombinant toxin A or B. If toxin ispresent in a specimen, this toxin will compete with the immobilizedrecombinant toxin protein for binding to the anti-recombinant antibodythereby reducing the signal obtained following the addition of areporter reagent. The reporter reagent detects the presence of antibodybound to the immobilized toxin protein.

In the second format, a sandwich immunoassay was developed usingaffinity-purified antibodies to recombinant toxin A and B. Theaffinity-purified antibodies to recombinant toxin A and B were used tocoat microtiter wells instead of the recombinant polypeptides (as wasdone in the competitive assay format). Biological samples containingtoxin A or B were then added to the wells followed by the addition of areporter reagent to detect the presence of bound toxin in the well.

a) Competitive Immunoassay for the Detection of C. difficile Toxin

Recombinant toxin A or B was attached to a solid support by coating 96well microtiter plates with the toxin protein at a concentration of 1μg/ml in PBS. The plates were incubated overnight at 2-8° C. Thefollowing morning, the coating solutions were removed and the remainingprotein binding sites on the wells were blocked by filling each wellwith a PBS solution containing 0.5% BSA and 0.05% Tween-20. Native C.difficile toxin A or B (Tech Lab) was diluted to 4 μg/ml in stoolextracts from healthy Syrian hamsters (Sasco). The stool extracts weremade by placing fecal pellets in a 15 ml centrifuge tube; PBS was addedat 2 ml/pellet and the tube was vortexed to create a uniform suspension.The tube was then centrifuged at 2000 rpm for 5 min at room temperature.The supernatant was removed; this comprises the stool extract. Fifty μlof the hamster stool extract was pipetted into each well of themicrotiter plates to serve as the diluent for serial dilutions of the 4μg/ml toxin samples. One hundred μl of the toxin samples at 4 μg/ml waspipetted into the first row of wells in the microtiter plate, and 50 μlaliquots were removed and diluted serially down the plate in duplicate.An equal volume of affinity purified anti-recombinant toxin antibodies[1 ng/well of anti-pMA1870-2680 antibody was used for the detection oftoxin A; 0.5 ng/well of anti-pMB1750-2360(Gerbu) was used for thedetection of toxin B] were added to appropriate wells, and the plateswere incubated at room temperature for 2 hours with gentle agitation.Wells serving as negative control contained antibody but no native toxinto compete for binding.

Unbound toxin and antibody were removed by washing the plates 3 to 5times with PBS containing 0.05% Tween-20. Following the wash step, 100μl of rabbit anti-chicken IgG antibody conjugated to alkalinephosphatase (Sigma) was added to each well and the plates were incubatedfor 2 hours at room temperature. The plates were then washed as beforeto remove unbound secondary antibody. Freshly prepared alkalinephosphatase substrate (1 mg/ml p-nitrophenyl phosphate (Sigma) in 50 mMNa₂CO₃, pH 9.5; 10 mM MgCl₂) was added to each well. Once sufficientcolor developed, the plates were read on a Dynatech MR700 microtiterplate reader using a 410 nm filter.

The results are summarized in Tables 32 and 33. For the results shown inTable 32, the wells were coated with recombinant toxin A protein(pMA1870-2680). The amount of native toxin A added (present as anaddition to solubilized hamster stool) to a given well is indicated (0to 200 ng). Antibody raised against the recombinant toxin A protein,pMA1870-2680, was affinity purified on the an affinity column containingpPA1870-2680 (described in Example 20). As shown in Table 32, therecombinant toxin A protein and affinity-purified antitoxin can be usedfor the basis of a competitive immunoassay for the detection of toxin Ain biological samples.

Similar results were obtained using the recombinant toxin B, pPB1750-2360, and antibodies raised against pMB1750-2360(Gerbu). For theresults shown in Table 33, the wells were coated with recombinant toxinB protein (pPB1750-2360). The amount of native toxin B added (present asan addition to solubilized hamster stool) to a given well is indicated(0 to 200 ng). Antibody raised against the recombinant toxin B protein,pMB1750-2360(Gerbu), was affinity purified on the an affinity columncontaining pPB 1850-2360 (described in Example 20). As shown in Table33, the recombinant toxin B protein and affinity-purified antitoxin canbe used for the basis of a competitive immunoassay for the detection oftoxin B in biological samples.

In this competition assay, the reduction is considered significant overthe background levels at all points; therefore the assay can be used todetect samples containing less than 12.5 ng toxin A/well and as littleas 50-100 ng toxin B/well. TABLE 32 Competitive Inhibition Of Anti-C.difficile Toxin A By Native Toxin A ng Toxin A/Well OD₄₁₀ Readout 2000.176 100 0.253 50 0.240 25 0.259 12.5 0.309 6.25 0.367 3.125 0.417 00.590

TABLE 33 Competitive Inhibition Of Anti-C. difficile Toxin B By NativeToxin B ng Toxin B/Well OD₄₁₀ Readout 200 0.392 100 0.566 50 0.607 250.778 12.5 0.970 6.25 0.902 3.125 1.040 0 1.055

These competitive inhibition assays demonstrate that native C. difficiletoxins and recombinant C. difficile toxin proteins can compete forbinding to antibodies raised against recombinant C. difficile toxinsdemonstrating that these anti-recombinant toxin antibodies provideeffective diagnostic reagents.

b) Sandwich Immunoassay for the Detection of C. difficile Toxin

Affinity-purified antibodies against recombinant toxin A or toxin B wereimmobilized to 96 well microtiter plates as follows. The wells werepassively coated overnight at 4° C. with affinity purified antibodiesraised against either pMA1870-2680 (toxin A) or pMB1750-2360(Gerbu)(toxin B). The antibodies were affinity purified as described in Example20. The antibodies were used at a concentration of 1 μg/ml and 100 μlwas added to each microtiter well. The wells were then blocked with 200μl of 0.5% BSA in PBS for 2 hours at room temperature and the blockingsolution was then decanted. Stool samples from healthy Syrian hamsterswere resuspended in PBS, pH 7.4 (2 ml PBS/stool pellet was used toresuspend the pellets and the sample was centrifuged as describedabove). The stool suspension was then spiked with native C. difficiletoxin A or B (Tech Lab) at 4 μg/ml. The stool suspensions containingtoxin (either toxin A or toxin B) were then serially diluted two-fold instool suspension without toxin and 50 μl was added in duplicate to thecoated microtiter wells. Wells containing stool suspension without toxinserved as the negative control.

The plates were incubated for 2 hours at room temperature and then werewashed three times with PBS. One hundred μl of either goat anti-nativetoxin A or goat anti-native toxin B (Tech Lab) diluted 1:1000 in PBScontaining 1% BSA and 0.05% Tween 20 was added to each well. The plateswere incubated for another 2 hours at room temperature. The plates werethen washed as before and 100 μl of alkaline phosphatase-conjugatedrabbit anti-goat IgG (Cappel, Durham, N.C.) was added at a dilution of1:1000. The plates were incubated for another 2 hours at roomtemperature. The plates were washed as before then developed by theaddition of 100 μl/well of a substrate solution containing 1 mg/mlp-nitrophenyl phosphate (Sigma) in 50 mM Na₂CO₃, pH 9.5; 10 mM MgCl₂.The absorbance of each well was measured using a plate reader (Dynatech)at 410 nm. The assay results are shown in Tables 34 and 35. TABLE 34 C.difficile Toxin A Detection In Stool Using Affinity- Purified AntibodiesAgainst Toxin A ng Toxin A/Well OD₄₁₀ Readout 200 0.9 100 0.8 50 0.73 250.71 12.5 0.59 6.25 0.421 0 0

TABLE 35 C. difficile Toxin B Detection In Stool Using Affinity-Purified Antibodies Against Toxin B ng Toxin A/Well OD₄₁₀ Readout 2001.2 100 0.973 50 0.887 25 0.846 12.5 0.651 6.25 0.431 0 0.004

The results shown in Tables 34 and 35 show that antibodies raisedagainst recombinant toxin A and toxin B fragments can be used to detectthe presence of C. difficile toxin in stool samples. These antibodiesform the basis for a sensitive sandwich immunoassay which is capable ofdetecting as little as 6.25 ng of either toxin A or B in a 50 μl stoolsample. As shown above in Tables 34 and 35, the background for thissandwich immunoassay is extremely low; therefore, the sensitivity ofthis assay is much lower than 6.25 ng toxin/well. It is likely thattoxin levels of 0.5 to 1.0 μg/well could be detected by this assay.

The results shown above in Tables 32-35 demonstrate clear utility of therecombinant reagents in C. difficile toxin detection systems.

Example 22 Construction and Expression of C. botulinum C Fragment FusionProteins

The C. botulinum type A neurotoxin gene has been cloned and sequenced[Thompson, et al., Eur. J. Biochem. 189:73 (1990)]. The nucleotidesequence of the toxin gene is available from the EMBL/GenBank sequencedata banks under the accession number X52066; the nucleotide sequence ofthe coding region is listed in SEQ ID NO:27. The amino acid sequence ofthe C. botulinum type A neurotoxin is listed in SEQ ID NO:28. The type Aneurotoxin gene is synthesized as a single polypeptide chain which isprocessed to form a dimer composed of a light and a heavy chain linkedvia disulfide bonds. The 50 kD carboxy-terminal portion of the heavychain is referred to as the C fragment or the H_(C) domain.

Previous attempts by others to express polypeptides comprising the Cfragment of C. botulinum type A toxin as a native polypeptide (e.g., notas a fusion protein) in E. coli have been unsuccessful [H.F.LaPenotiere, et al. in Botulinum and Tetanus Neurotoxins, DasGupta, Ed.,Plenum Press, New York (1993), pp. 463-466]. Expression of the Cfragment as a fusion with the E. coli MBP was reported to result in theproduction of insoluble protein (H. F. LaPenotiere, et al., supra).

In order to produce soluble recombinant C fragment proteins in E. coli,fusion proteins comprising a synthetic C fragment gene derived from theC. botulinum type A toxin and either a portion of the C. difficile toxinprotein or the MBP were constructed. This example involved a) theconstruction of plasmids encoding C fragment fusion proteins and b)expression of C. botulinum C fragment fusion proteins in E. coli.

a) Construction of Plasmids Encoding C Fragment Fusion Proteins

In Example 11, it was demonstrated that the C. difficile toxin A repeatdomain can be efficiently expressed and purified in E. coli as eithernative (expressed in the pET 23a vector in clone pPA1870-2680) or fusion(expressed in the pMALc vector as a fusion with the E. coli MBP in clonepMA1870-2680) proteins. Fusion proteins comprising a fusion between theMBP, portions of the C. difficile toxin A repeat domain (shown to beexpressed as a soluble fusion protein) and the C fragment of the C.botulinum type A toxin were constructed. A fusion protein comprising theC fragment of the C. botulinum type A toxin and the MBP was alsoconstructed.

FIG. 25 provides a schematic representation of the botulinal fusionproteins along with the donor constructs containing the C. difficiletoxin A sequences or C. botulinum C fragment sequences which were usedto generate the botulinal fusion proteins. In FIG. 25, the solid boxesrepresent C. difficile toxin A gene sequences, the open boxes representC. botulinum C fragment sequences and the solid black ovals representthe E. coli MBP. When the name for a restriction enzyme appears insideparenthesis, this indicates that the restriction site was destroyedduring construction. An asterisk appearing with the name for arestriction enzyme indicates that this restriction site was recreated atthe cloning junction.

In FIG. 25, a restriction map of the pMA1870-2680 and pPA1100-2680constructs (described in Example 11) which contain sequences derivedfrom the C. difficile toxin A repeat domain are shown; these constructswere used as the source of C. difficile toxin A gene sequences for theconstruction of plasmids encoding fusions between the C. botulinum Cfragment gene and the C. difficile toxin A gene. The pMA1870-2680expression construct expresses high levels of soluble, intact fusionprotein (20 mg/liter culture) which can be affinity purified on anamylose column (purification described in Example 11d).

The pAlterBot construct (FIG. 25) was used as the source of C. botulinumC fragment gene sequences for the botulinal fusion proteins. pAlterBotwas obtained from J. Middlebrook and R. Lemley at the U.S. Department ofDefense. pAlterBot contains a synthetic C. botulinum C fragment insertedin to the pALTER-1® vector (Promega). This synthetic C fragment geneencodes the same amino acids as does the naturally occurring C fragmentgene. The naturally occurring C fragment sequences, like mostclostridial genes, are extremely A/T rich (Thompson et al., supra). Thishigh A/T content creates expression difficulties in E. coli and yeastdue to altered codon usage frequency and fortuitous polyadenylationsites, respectively. In order to improve the expression of C fragmentproteins in E. coli, a synthetic version of the gene was created inwhich the non-preferred codons were replaced with preferred codons.

The nucleotide sequence of the C. botulinum C fragment gene sequencescontained within pAlterBot is listed in SEQ ID NO:22. The first sixnucleotides (ATGGCT) encode a methionine and alanine residue,respectively. These two amino acids result from the insertion of the C.botulinum C fragment sequences into the pALTER® vector and provide theinitiator methionine residue. The amino acid sequence of the C.botulinum C fragment encoded by the sequences contained within pAlterBotis listed in SEQ ID NO:23. The first two amino acids (Met Ala) areencoded by vector-derived sequences. From the third amino acid residueonward (Arg), the amino acid sequence is identical to that found in theC. botulinum type A toxin gene.

The pMA1870-2680, pPA1100-2680 and pAlterBot constructs were used asprogenitor plasmids to make expression constructs in which fragments ofthe C. difficile toxin A repeat domain were expressed as genetic fusionswith the C. botulinum C fragment gene using the pMAL-c expression vector(New England BioLabs). The pMAL-c expression vector generates fusionproteins which contain the MBP at the amino-terminal end of the protein.A construct, pMBot, in which the C. botulinum C fragment gene wasexpressed as a fusion with only the MBP was constructed (FIG. 25).Fusion protein expression was induced from E. coli strains harboring theabove plasmids, and induced protein was affinity purified on an amyloseresin column.

i) Construction of pBlueBot

In order to facilitate the cloning of the C. botulinum C fragment genesequences into a number of desired constructs, the botulinal genesequences were removed from pAlterBot and were inserted into thepBluescript plasmid (Stratagene) to generate pBlueBot (FIG. 25).pBlueBot was constructed as follows. Bacteria containing the pAlterBotplasmid were grown in medium containing tetracycline and plasmid DNA wasisolated using the QIAprep-spin Plasmid Kit (Qiagen). One microgram ofpAlterBot DNA was digested with NcoI and the resulting 3′ recessedsticky end was made blunt using the Klenow fragment of DNA polymerase I(here after the Klenow fragment). The pAlterBot DNA was then digestedwith HindIII to release the botulinal gene sequences (the Bot insert) asa blunt (filled NcoI site)-HindIII fragment. pBluescript vector DNA wasprepared by digesting 200 ng of pBluescript DNA with SmaI and HindIII.The digestion products from both plasmids were resolved on an agarosegel. The appropriate fragments were removed from the gel, mixed andpurified utilizing the Prep-a-Gene kit (BioRad). The eluted DNA was thenligated using T4 DNA ligase and used to transform competent DH5a cells(Gibco-BRL). Host cells were made competent for transformation using thecalcium chloride protocol of Sanbrook et al., supra at 1.82-1.83.Recombinant clones were isolated and confirmed by restriction digestionusing standard recombinant molecular biology techniques (Sambrook et al,supra). The resultant clone, pBlueBot, contains several useful uniquerestriction sites flanking the Bot insert (i.e., the C. botulinum Cfragment sequences derived from pAlterBot) as shown in FIG. 25.

ii) Construction of C. difficile/C. botulinum/MBP Fusion Proteins

Constructs encoding fusions between the C. difficile toxin A gene andthe C. botulinum C fragment gene and the MBP were made utilizing thesame recombinant DNA methodology outlined above; these fusion proteinscontained varying amounts of the C. difficile toxin A repeat domain.

The pMABot clone contains a 2.4 kb insert derived from the C. difficiletoxin A gene fused to the Bot insert (i.e, the C. botulinum C fragmentsequences derived from pAlterBot). pMABot (FIG. 25) was constructed bymixing gel-purified DNA from NotI/HindIII digested pBlueBot (the 1.2 kbBot fragment), SpeI/NotI digested pPA1100-2680 (the 2.4 kb C. difficiletoxin A repeat fragment) and XbaI/HindIII digested pMAL-c vector.Recombinant clones were isolated, confirmed by restriction digestion andpurified using the QIAprep-spin Plasmid Kit (Qiagen). This cloneexpresses the toxin A repeats and the botulinal C fragment proteinsequences as an in-frame fusion with the MBP.

The pMCABot construct contains a 1.0 kb insert derived from the C.difficile toxin A gene fused to the Bot insert (i.e, the C. botulinum Cfragment sequences derived from pAlterBot). pMCABot was constructed bydigesting the pMABot clone with EcoRI to remove the 5′ end of the C.difficile toxin A repeat (see FIG. 25, the pMAL-c vector contains aEcoRI site 5′ to the C. difficile insert in the pMABot clone). Therestriction sites were filled and religated together after gelpurification. The resultant clone (pMCABot, FIG. 25) generated anin-frame fusion between the MBP and the remaining 3′ portion of the C.difficile toxin A repeat domain fused to the Bot gene.

The pMNABot clone contains the 1 kb SpeI/EcoRI (filled) fragment fromthe C. difficile toxin A repeat domain (derived from clone pPA1100-2680) and the 1.2 kb C. botulinum C fragment gene as a NcoI(filled)/HindIII fragment (derived from pAlterBot). These two fragmentswere inserted into the pMAL-c vector digested with XbaI/HindIII. The twoinsert fragments were generated by digestion of the appropriate plasmidwith EcoRI (pPA1100-2680) or NcoI (pAlterBot) followed by treatment withthe Klenow fragment. After treatment with the Klenow fragment, theplasmids were digested with the second enzyme (either SpeI or HindIII).All three fragments were gel purified, mixed and Prep-a-Gene purifiedprior to ligation. Following ligation and transformation, putativerecombinants were analyzed by restriction analysis; the EcoRI site wasfound to be regenerated at the fusion junction, as was predicted for afusion between the filled EcoRI and NcoI sites.

A construct encoding a fusion protein between the botulinal C fragmentgene and the MBP gene was constructed (i.e., this fusion lacks any C.difficile toxin A gene sequences) and termed pMBot. The pMBot-constructwas made by removal of the C. difficile toxin A sequences from thepMABot construct and fusing the C fragment gene sequences to the MBP.This was accomplished by digestion of pMABot DNA with StuI (located inthe pMALc polylinker 5′ to the XbaI site) and XbaI (located 3′ to theNotI site at the toxA-Bot fusion junction), filling in the XbaI siteusing the Klenow fragment, gel purifying the desired restrictionfragment, and ligating the blunt ends to circularize the plasmid.Following ligation and transformation, putative recombinants wereanalyzed by restriction mapping of the Bot insert (i.e, the C. botulinumC fragment sequences).

b) Expression of C. botulinum C Fragment Fusion Proteins in E. coli

Large scale (1 liter) cultures of the pMAL-c vector, and eachrecombinant construct described above in (a) were grown, induced, andsoluble protein fractions were isolated as described in Example 18. Thesoluble protein extracts were chromatographed on amylose affinitycolumns to isolate recombinant fusion protein. The purified recombinantfusion proteins were analyzed by running samples on SDS-PAGE gelsfollowed by Coomassie staining and by Western blot analysis as described[Williams et al, (1994) supra]. In brief, extracts were prepared andchromatographed in column buffer (10 mM NaPO₄, 0.5 M NaCl, 10 mMβ-mercaptoethanol, pH 7.2) over an amylose resin (New England Biolabs)column, and eluted with column buffer containing 10 mM maltose asdescribed [Williams, et al. (1994), supra]. An SDS-PAGE gel containingthe purified protein samples stained with Coomassie blue is shown inFIG. 26.

In FIG. 26, the following samples were loaded. Lanes 1-6 contain proteinpurified from E. coli containing the pMAL-c, pPA1870-2680, pMABot,pMNABot, pMCABot and pMBot plasmids, respectively. Lane 7 contains broadrange molecular weight protein markers (BioRad).

The protein samples were prepared for electrophoresis by mixing 5 μl ofeluted protein with 5 μl of 2×SDS-PAGE sample buffer (0.125 mM Tris-HCl,pH 6.8, 2 mM EDTA, 6% SDS, 20% glycerol, 0.025% bromophenol blue;L-mercaptoethanol is added to 5% before use). The samples were heated to95° C. for 5 min, then cooled and loaded on a 7.5% agarose SDS-PAGE gel.Broad range molecular weight protein markers were also loaded to allowestimation of the MW of identified fusion proteins. Afterelectrophoresis, protein was detected generally by staining the gel withCoomassie blue.

In all cases the yields were in excess of 20 mg fusion protein per literculture (see Table 36) and, with the exception of the pMCABot protein, ahigh percentage (i.e., greater than 20-50% of total eluted protein) ofthe eluted fusion protein was of a MW predicted for the full lengthfusion protein (FIG. 26). It was estimated (by visual inspection) thatless than 10% of the pMCABot fusion protein was expressed as the fulllength fusion protein. TABLE 36 Yield Of Affinity Purified C. botulinumC Fragment/MBP Fusion Proteins Yield (mg/liter of Percentage Of TotalConstruct Culture) Soluble Protein pMABot 24 5.0 pMCABot 34 5.0 pMNABot40 5.5 pMBot 22 5.0 pMA1870-2680 40 4.8

These results demonstrate that high level expression of intact C.botulinum C fragment/C. difficile toxin A fusion proteins in E. coli isfeasible using the pMAL-c expression system. These results are incontrast to those reported by H. F. LaPenotiere, et al. (1993), supra.In addition, these results show that it is not necessary to fuse thebotulinal C fragment gene to the C. difficile toxin A gene in order toproduce a soluble fusion protein using the pMAL-c system in E. coli.

In order to determine whether the above-described botulinal fusionproteins were recognized by anti-C. botulinum toxin A antibodies,Western blots were performed. Samples containing affinity-purifiedproteins from E. coli containing the pMABot, pMCABot, pMNABot, pMBot,pMA1870-2680 or pMALc plasmids were analyzed. SDS-PAGE gels (7.5%acrylamide) were loaded with protein samples purified from eachexpression construct. After electrophoresis, the gels were blotted andprotein transfer was confirmed by Ponceau S staining (as described inExample 12b).

Following protein transfer, the blots were blocked by incubation for 1hr at 20° C. in blocking buffer [PBST (PBS containing 0.1% Tween 20 and5% dry milk)]. The blots were then incubated in 10 ml of a solutioncontaining the primary antibody; this solution comprised a 1/500dilution of an anti-C. botulinum toxin A IgY PEG prep (described inExample 3) in blocking buffer. The blots were incubated for 1 hr at roomtemperature in the presence of the primary antibody. The blots werewashed and developed using a rabbit anti-chicken alkaline phosphataseconjugate (Boehringer ManNheIm) as the secondary antibody as follows.The rabbit anti-chicken antibody was diluted to 1 μg/ml in blockingbuffer (10 ml final volume per blot) and the blots were incubated atroom temperature for 1 hour in the presence of the secondary antibody.The blots were then washed successively with PBST, BBS-Tween and 50 mMNa₂CO₃, pH 9.5. The blots were then developed in freshly-preparedalkaline phosphatase substrate buffer (100 μg/ml nitro blue tetrazolium,50 μg/ml 5-bromo-chloro-indolylphosphate, 5 mM MgCl₂ in 50 mM Na₂CO₃, pH9.5). Development was stopped by flooding the blots with distilled waterand the blots were air dried.

This Western blot analysis detected anti-C. botulinum toxin reactiveproteins in the pMABot, pMCABot, pMNABot and pMBot protein samples(corresponding to the predicted full length proteins identified above byCoomassie staining in FIG. 26), but not in the pMA1100-2680 or pMALcprotein samples.

These results demonstrate that the relevant fusion proteins purified onan amylose resin as described above in section a) containedimmunoreactive C. botulinum C fragment protein as predicted.

Example 23 Generation of Neutralizing Antibodies by Nasal Administrationof pMBot Protein

The ability of the recombinant botulinal toxin proteins produced inExample 22 to stimulate a systemic immune response against botulinaltoxin epitopes was assessed. This example involved: a) the evaluation ofthe induction of serum IgG titers produced by nasal or oraladministration of botulinal toxin-containing C. difficile toxin A fusionproteins and b) the in vivo neutralization of C. botulinum type Aneurotoxin by anti-recombinant C. botulinum C fragment antibodies.

a) Evaluation of the Induction of Serum IgG Titers Produced by Nasal orOral Administration of Botulinal Toxin-Containing C. difficile Toxin AFusion Proteins

Six groups containing five 6 week old CF female rats (Charles River) pergroup were immunized nasally or orally with one of the following threecombinations using protein prepared in Example 22: (1) 250 μg pMBotprotein per rat (nasal and oral); 2) 250 μg pMABot protein per rat(nasal and oral); 3) 125 μg pMBot admixed with 125 μg pMA1870-2680 perrat (nasal and oral); A second set of 5 groups containing 3 CF femalerats/group were immunized nasally or orally with one of the followingcombinations (4) 250 μg pMNABot protein per rat (nasal and oral) or 5)250 μg pMAL-c protein per rat (nasal and oral).

The fusion proteins were prepared for immunization as follows. Theproteins (in column buffer containing 10 mM maltose) were diluted in 0.1M carbonate buffer, pH 9.5 and administered orally or nasally in a 200μl volume. The rats were lightly sedated with ether prior toadministration. The oral dosing was accomplished using a 20 gaugefeeding needle. The nasal dosing was performed using a P-200micro-pipettor (Gilson). The rats were boosted 14 days after the primaryimmunization using the techniques described above and were bled 7 dayslater. Rats from each group were lightly etherized and bled from thetail. The blood was allowed to clot at 37° C. for 1 hr and the serum wascollected.

The serum from individual rats was analyzed using an ELISA to determinethe anti-C. botulinum type A toxin IgG serum titer. The ELISA protocolused is a modification of that described in Example 13c. Briefly,96-well microtiter plates (Falcon, Pro-Bind Assay Plates) were coatedwith C. botulinum type A toxoid (prepared as described in Example 3a) byplacing 100 μl volumes of C. botulinum type A toxoid at 2.5 μg/ml in PBScontaining 0.005% thimerosal in each well and incubating overnight at 4°C. The next morning, the coating suspensions were decanted and all wellswere washed three times using PBS.

In order to block non-specific binding sites, 100 μl of blockingsolution [0.5% BSA in PBS] was then added to each well and the plateswere incubated for 1 hr at 37° C. The blocking solution was decanted andduplicate samples of 150 μl of diluted rat serum added to the first wellof a dilution series. The initial testing serum dilution was 1:30 inblocking solution containing 0.5% Tween 20 followed by 5-fold dilutionsinto this solution. This was accomplished by serially transferring 30 μlaliquots to 120 μl blocking solution containing 0.5% Tween 20, mixing,and repeating the dilution into a fresh well. After the final dilution,30 μl was removed from the well such that all wells contained 120 μlfinal volume. A total of 3 such dilutions were performed (4 wellstotal). The plates were incubated 1 hr at 37° C. Following thisincubation, the serially diluted samples were decanted and the wellswere washed six times using PBS containing 0.5% Tween 20 (PBST). To eachwell, 100 μl of a rabbit anti-Rat IgG alkaline phosphatase (Sigma)diluted 1/1000 in blocking buffer containing 0.5% Tween 20 was added andthe plate was incubated for 1 hr at 37° C. The conjugate solutions weredecanted and the plates were washed as described above, substituting 50mM Na₂CO₃, pH 9.5 for the PBST in the final wash. The plates weredeveloped by the addition of −100 μl of a solution containing 1 mg/mlpara-nitro phenyl phosphate (Sigma) dissolved in 50 mM Na₂CO₃, 10 mMMgCl₂, pH 9.5 to each well, and incubating the plates at roomtemperature in the dark for 5-45 min. The absorbency of each well wasmeasured at 410 mm using a Dynatech MR 700 plate reader. The results aresummarized in Tables 37 and 38 and represent mean serum reactivities ofindividual mice. TABLE 37 Determination Of Anti-C. botulinum Type AToxin Serum IgG Titers Following Immunization With C. botulinum CFragment- Containing Fusion Proteins Nasal Oral Route of ImmunizationpMBot & pMBot & Immunogen PREIMMUNE pMBot pMA1870-2680 pMABot pMBotpMA1870-2680 pMABot Dilution 1:30 0.080 1.040 1.030 0.060 0.190 0.0800.120 1:150 0.017 0.580 0.540 0.022 0.070 0.020 0.027 1:750 0.009 0.2800.260 0.010 0.020 0.010 0.014 1:3750 0.007 0.084 0.090 0.009 0.009 0.0100.007 # Rats Tested 5 5 5 5 2 2*Numbers represent the average values obtained from two ELISA plates,standardized utilizing the preimmune control.

TABLE 38 Determination Of Anti-C. botulinum Type A Toxin Serum IgGTiters Following Immunization With C. botulinum C Fragment- ContainingFusion Proteins Route of Immunization Nasal Oral Immunogen PREIMMUNEpMBot pMABot pMBot pMABot Dilution 1:30 0.040 1.557 1.010 0.015 0.0101:150 0.009 0.383 0.0001 0.003 0.002 1:750 0.001 0.140 0.000 0.000 0.0001:3750 0.000 0.040 0.000 0.000 0.000 # 1 1 3 3 Rats Tested

The above ELISA results demonstrate that reactivity against thebotulinal fusion proteins was strongest when the route of administrationwas nasal; only weak responses were stimulated when the botulinal fusionproteins were given orally. Nasally delivered pMbot and pMBot admixedwith pMA1870-2680 invoked the greatest serum IgG response. These resultsshow that only the pMBot protein is necessary to induce this response,since the addition of the pMA1870-2680 protein did not enhance antibodyresponse (Table 37). Placement of the C. difficile toxin A fragmentbetween the MBP and the C. botulinum C fragment protein dramaticallyreduced anti-bot IgG titer (see results using pMABot, pMCABot andpMNABot proteins).

This study demonstrates that the pMBot protein induces a strong serumIgG response directed against C. botulinum type A toxin when nasallyadministered.

b) In Vivo Neutralization of C. botulinum Type A Neurotoxin byAnti-Recombinant C. botulinum C Fragment Antibodies

The ability of the anti-C. botulinum type A toxin antibodies generatedby nasal administration of recombinant botulinal fusion proteins in rats(Example 22) to neutralize C. botulinum type A toxin was tested in amouse neutralization model. The mouse model is the art accepted methodfor detection of botulinal toxins in body fluids and for the evaluationof anti-botulinal antibodies [E. J. Schantz and D. A. Kautter, J. Assoc.Off. Anal. Chem. 61:96 (1990) and Investigational New Drug (BB-IND-3703)application by the Surgeon General of the Department of the Army to theFederal Food and Drug Administration]. The anti-C. botulinum type Atoxin antibodies were prepared as follows.

Rats from the group given pMBot protein by nasal administration wereboosted a second time with 250 μg pMBot protein per rat and serum wascollected 7 days later. Serum from one rat from this group and from apreimmune rat was tested for anti-C. botulinum type A toxin neutralizingactivity in the mouse neutralization model described below.

The LD₅₀ of a solution of purified C. botulinum type A toxin complex,obtained from Dr. Eric Johnson (University of Wisconsin Madison), wasdetermined using the intraperitoneal (IP) method of Schantz and Kautter[J. Assoc. Off. Anal. Chem. 61:96 (1978)] using 18-22 gram female ICRmice and was found to be 3500 LD₅₀/ml. The determination of the LD₅₀ wasperformed as follows. A Type A toxin standard was prepared by dissolvingpurified type A toxin complex in 25 mM sodium phosphate buffer, pH 6.8to yield a stock toxin solution of 3.15×10⁷ LD₅₀/mg. The OD₂₇₈ of thesolution was determined and the concentration was adjusted to 10-20μg/ml. The toxin solution was then diluted 1:100 in gel-phosphate (30 mMphosphate, pH 6.4; 0.2% gelatin). Further dilutions of the toxinsolution were made as shown below in Table 39. Two mice were injected IPwith 0.5 ml of each dilution shown and the mice were observed forsymptoms of botulism for a period of 72 hours. TABLE 39 Determination OfThe LD₅₀ Of Purified C. botulinum Type A Toxin Complex Dilution NumberDead At 72 hr 1:320 2/2 1:640 2/2 1:1280 2/2 1:2560 0/2 (sick after 72hr) 1:5120 0/2 (no symptoms)

From the results shown in Table 39, the toxin titer was assumed to bebetween 2560 LD₅₀/ml and 5120 LD₅₀/ml (or about 3840 LD₅₀/ml). Thisvalue was rounded to 3500 LD₅₀/ml for the sake of calculation.

The amount of neutralizing antibodies present in the serum of ratsimmunized nasally with pMBot protein was then determined. Serum from tworats boosted with pMBot protein as described above and preimmune serumfrom one rat was tested as follows. The toxin standard was diluted 1:100in gel-phosphate to a final concentration of 350 LD₅₀/ml. One milliliterof the diluted toxin standard was mixed with 25 μl of serum from each ofthe three rats and 0.2 ml of gel-phosphate. The mixtures were incubatedat room temperature for 30 min with occasional mixing. Each of two micewere injected with IP with 0.5 ml of the mixtures. The mice wereobserved for signs of botulism for 72 hr. Mice receiving serum from ratsimmunized with pMBot protein neutralized this challenge dose. Micereceiving preimmune rat serum died in less than 24 hr.

The amount of neutralizing anti-toxin antibodies present in the serum ofrats immunized with pMBot protein was then quantitated. Serum antibodytitrations were performed by mixing 0.1 ml of each of the antibodydilutions (see Table 40) with 0.1 ml of a 1:10 dilution of stock toxinsolution (3.5×10⁴ LD₅₀/ml) with 1.0 ml of gel-phosphate and injecting0.5 ml IP into 2 mice per dilution. The mice were then observed forsigns of botulism for 3 days (72 hr). The results are tabulated in Table39.

As shown in Table 40 pMBot serum neutralized C. botulinum type A toxincomplex when used at a dilution of 1:320 or less. A mean neutralizingvalue of 168 IU/ml was obtained for the pMBot serum (an IU is defined as10,000 mouse LD₅₀). This value translates to a circulating serum titerof about 3.7 IU/mg of serum protein. This neutralizing titer iscomparable to the commercially available bottled concentrated (ConnaughtLaboratories, Ltd.) horse anti-C. botulinum antiserum. A 10 ml vial ofConnaught antiserum contains about 200 mg/ml of protein; each ml canneutralize 750 IU of C. botulinum type A toxin. After administration ofone vial to a human, the circulating serum titer of the Connaughtpreparation would be approximately 25 IU/ml assuming an average serumvolume of 3 liters). Thus, the circulating anti-C. botulinum titer seenin rats nasally immunized with pMBot protein (168 IU/ml) is 6.7 timehigher than the necessary circulation titer of anti-C. botulinumantibody needed to be protective in humans. TABLE 40 Quantitation OfNeutralizing Antibodies In pMBot Sera pMBot^(a) Dilution Rat 1 Rat 21:20 2/2 2/2 1:40 2/2 2/2 1:80 2/2 2/2 1:160 2/2 2/2 1:320 2/2^(b)2/2^(b) 1:640 0/2 0/2 1:1280 0/2 0/2 1:2560 0/2 0/2^(a)Numbers represent the number of mice surviving at 72 hours whichreceived serum taken from rats immunized with the pMBot protein.^(b)These mice survived but were sick after 72 hr.

These results demonstrate that antibodies capable of neutralizing C.botulinum type A toxin are induced when recombinant C. botulinum Cfragment fusion protein produced in E. coli is used as an immunogen.

Example 24 Production of Soluble C. botulinum C Fragment ProteinSubstantially Free of Endotoxin Contamination

Example 23 demonstrated that neutralizing antibodies are generated byimmunization with the pMBot protein expressed in E. coli. These resultsshowed that the pMBot fusion protein is a good vaccine candidate.However, immunogens suitable for use as vaccines should be pyrogen-freein addition to having the capability of inducing neutralizingantibodies. Expression clones and conditions that facilitate theproduction of C. botulinum C fragment protein for utilization as avaccine were developed.

The example involved: (a) determination of pyrogen content of the pMBotprotein; (b) generation of C. botulinum C fragment protein free of theMBP; (c) expression of C. botulinum C fragment protein using variousexpression vectors; and (d) purification of soluble C. botulinum Cfragment protein substantially free of significant endotoxincontamination.

a) Determination of the Pyrogen Content of the pMBot Protein

In order to use a recombinant antigen as a vaccine in humans or otheranimals, the antigen preparation must be shown to be free of pyrogens.The most significant pyrogen present in preparations of recombinantproteins produced in gram-negative bacteria, such as E. coli, isendotoxin [F. C. Pearson, Pyrogens: endotoxins, LAL testing anddepyrogenation, (1985) Marcel Dekker, New York, pp. 23-56]. To evaluatethe utility of the pMBot protein as a vaccine candidate, the endotoxincontent in MBP fusion proteins was determined.

The endotoxin content of recombinant protein samples was assayedutilizing the Limulus assay (LAL kit; Associates of Cape Cod) accordingto the manufacturer's instructions. Samples of affinity-purified pMa1-cprotein and pMA1870-2680 were found to contain high levels of endotoxin[>50,000 EU/mg protein; EU (endotoxin unit)]. This suggested that MBP-or toxin A repeat-containing fusions with the botulinal C fragmentshould also contain high levels of endotoxin. Accordingly, removal ofendotoxin from affinity-purified pMa1-c and pMBot protein preparationswas attempted as follows.

Samples of pMa1-c and pMBot protein were depyrogenated with polymyxin todetermine if the endotoxin could be easily removed. The following amountof protein was treated: 29 ml at 4.8 OD₂₈₀/ml for pMa1-c and 19 mls at1.44 OD₂₈₀ ml for pMBot. The protein samples were dialyzed extensivelyagainst PBS and mixed in a 50 ml tube (Falcon) with 0.5 mlPBS-equilibrated polymyxin B (Affi-Prep Polymyxin, BioRad). The sampleswere allowed to mix by rotating the tubes overnight at 4° C. Thepolymyxin was pelleted by centrifugation for 30 min in a bench topcentrifuge at maximum speed (approximately 2000×g) and the supernatantwas removed. The recovered protein (in the supernatant) was quantifiedby OD₂₈₀, and the endotoxin activity was assayed by LAL. In both casesonly approximately ⅓ of the input protein was recovered and thepolymyxin-treated protein retained significant endotoxin contamination(approximately 7000 EU/mg of pMBot).

The depyrogenation experiment was repeated using an independentlypurified pMa1-c protein preparation and similar results were obtained.From these studies it was concluded that significant levels of endotoxincopurifies with these MBP fusion proteins using the amylose resin.Furthermore, this endotoxin cannot be easily removed by polymyxintreatment.

These results suggest that the presence of the MBP sequences on thefusion protein complicated the removal of endotoxin from preparations ofthe pMBot protein.

b) Generation of C. botulinum C Fragment Protein Free of the MBP

It was demonstrated that the pMBot fusion protein could not be easilypurified from contaminating endotoxin in section a) above. The abilityto produce a pyrogen-free (e.g., endotoxin-free) preparation of solublebotulinal C fragment protein free of the MBP tag was next investigated.The pMBot expression construct was designed to facilitate purificationof the botulinal C fragment from the MBP tag by cleavage of the fusionprotein by utilizing an engineered FactorXa cleavage site presentbetween the MBP and the botulinal C fragment. The FactorXa cleavage wasperformed as follows.

FactorXa (New England Biolabs) was added to the pMBot protein (using a0.1-1.0% FactorXa/pMBot protein ratio) in a variety of buffer conditions[e.g., PBS-NaCl (PBS containing 0.5 M NaCl), PBS-NaCl containing 0.2%Tween 20, PBS, PBS containing 0.2% Tween 20, PBS-C (PBS containing 2 mMCaCl₂), PBS-C containing either 0.1 or 0.5% Tween 20, PBS-C containingeither 0.1 or 0.5% NP-40, PBS-C containing either 0.1 or 0.5% TritonX-100, PBS-C containing 0.1% sodium deoxycholate, PBS-C containing 0.1%SDS]. The FactorXa digestions were incubated for 12-72 hrs at roomtemperature.

The extent of cleavage was assessed by Western blot or Coomassie bluestaining of proteins following electrophoresis on denaturing SDS-PAGEgels, as described in Example 22. Cleavage reactions (and controlsamples of uncleaved pMBot protein) were centrifuged for 2 min in amicrofuge to remove insoluble protein prior to loading the samples onthe gel. The FactorXa treated samples were compared with uncleaved,uncentrifuged pMBot samples on the same gel. The results of thisanalysis is summarized below.

1) Most (about 90%) pMBot protein could be removed by centrifugation,even when uncleaved control samples were utilized. This indicated thatthe pMBot fusion protein was not fully soluble (i.e., it exists as asuspension rather than as a solution). [This result was consistent withthe observation that most affinity-purified pMBot protein precipitatesafter long term storage (>2 weeks) at 4° C. Additionally, the majority(i.e., 75%) of induced pMBot protein remains in the pellet aftersonication and clarification of the induced E. coli. Resuspension ofthese insoluble pellets in PBS followed by sonication results in partialsolubilization of the insoluble pMBot protein in the pellets.]

2) The portion of pMBot protein that is fully in solution (about 10% ofpMBot protein) is completely cleaved by FactorXa, but the cleaved(released) botulinal C fragment is relatively insoluble such that onlythe cleaved MBP remains fully in solution.

3) None of the above reaction conditions enhanced solubility withoutalso reducing-effective cleavage. Conditions that effectivelysolubilized the cleaved botulinal C fragment were not identified.

4) The use of 0.1% SDS in the buffer used for FactorXa cleavage enhancedthe solubility of the pMBot protein (all of pMBot protein was soluble).However, the presence of the SDS prevented any cleavage of the fusionprotein with FactorXa.

5) Analysis of pelleted protein from the cleavage reactions indicatedthat both full length pMBot (i.e., uncleaved) and cleaved botulinal Cfragment protein precipitated during incubation.

These results demonstrate that purification of soluble botulinal Cfragment protein after cleavage of the pMBot fusion protein iscomplicated by the insolubility of both the pMBot protein and thecleaved botulinal C fragment protein.

c) Expression of C. botulinum C Fragment Using Various ExpressionVectors

In order to determine if the solubility of the botulinal C fragment wasenhanced by expressing the C fragment protein as a native protein, anN-terminal His-tagged protein or as a fusion withglutathione-S-transferase (GST), alternative expression plasmids wereconstructed. These expression constructs were generated utilizing themethodologies described in Example 22. FIG. 27 provides a schematicrepresentation of the vectors described below.

In FIG. 27, the following abbreviations are used. pP refers to the pET23vector. pHIS refers to the pETHisa vector. pBlue refers to thepBluescript vector. pM refers to the pMAL-c vector and pG refers to thepGEX3T vector (described in Example 11). The solid black lines representC. botulinum C fragment gene sequences; the solid black ovals representthe MBP; the hatched ovals represent GST; “HHHHH” represents thepoly-histidine tag. In FIG. 27, when the name for a restriction enzymeappears inside parenthesis, this indicates that the restriction site wasdestroyed during construction. An asterisk appearing with the name for arestriction enzyme indicates that this restriction site was recreated ata cloning junction.

i) Construction of pPBot

In order to express the C. botulinum C fragment as a native (i.e.,non-fused) protein, the pPBot plasmid (shown schematically in FIG. 27)was constructed as follows. The C fragment sequences present inpAlterBot (Example 22) were removed by digestion of pAlterBot with NcoIand HindIII. The NcoI/HindIII C fragment insert was ligated to pETHisavector (described in Example 18b) which was digested with NcoI andHindIII. This ligation creates an expression construct in which theNcoI-encoded methionine of the botulinal C fragment is the initiatorcodon and directs expression of the native botulinal C fragment. Theligation products were used to transform competent BL21(DE3)pLysS cells(Novagen). Recombinant clones were identified by restriction mapping.

ii) Construction of pHisBot

In order to express the C. botulinum C fragment containing apoly-histidine tag at the amino-terminus of the recombinant protein, thepHisBot plasmid (shown schematically in FIG. 27) was constructed asfollows. The NcoI/HindIII botulinal C fragment insert from pAlterbot wasligated into the pETHisa vector which was digested with NheI andHindIII. The NcoI (on the C fragment insert) and NheI (on the pETHisavector) sites were filled in using the Klenow fragment prior toligation; these sites were then blunt end ligated (the NdeI site wasregenerated at the clone junction as predicted). The ligation productswere used to transform competent BL21(DE3)pLysS cells and recombinantclones were identified by restriction mapping.

The resulting pHisBot clone expresses the botulinal C fragment proteinwith a histidine-tagged N-terminal extension having the followingsequence: MetGlyH is H isHisHisHisHisHisHisHisHisSerSerGlyHisIleGluGlyArgHisMetAla (SEQ IDNO:24); the amino acids encoded by the botulinal C fragment gene areunderlined and the vector encoded amino acids are presented in plaintype. The nucleotide sequence present in the pETHisa vector whichencodes the pHisBot fusion protein is listed in SEQ ID NO:25. The aminoacid sequence of the pHisBot protein is listed in SEQ ID NO:26.

iii) Construction of pGBot

The botulinal C fragment protein was expressed as a fusion with theglutathione-S-transferase protein by constructing the pGBot plasmid(shown schematically in FIG. 27). This expression construct was createdby cloning the NotI/SalI C fragment insert present in pBlueBot (Example22) into the pGEX3T vector which was digested with SmaI and XhoI. TheNotI site (present on the botulinal fragment) was made blunt prior toligation using the Klenow fragment. The ligation products were used totransform competent BL21 cells.

Each of the above expression constructs were tested by restrictiondigestion to confirm the integrity of the constructs.

Large scale (1 liter) cultures of pPBot [BL21(DE3)pLysS host], pHisBot[BL21(DE3)pLysS host] and pGBot (BL21 host) were grown in 2X YT mediumand induced (using IPTG to 0.8-1.0 mM) for 3 hrs as described in Example22. Total, soluble and insoluble protein preparations were prepared from1 ml aliquots of each large scale culture [Williams et al. (1994),supra] and analyzed by SDS-PAGE. No obvious induced band was detectablein the pPBot or pHisBot samples by Coomassie staining, while a prominentinsoluble band of the anticipated MW was detected in the pGBot sample.Soluble lysates of the pGBot large scale (resuspended in PBS) or pHisBotlarge scale [resuspended in Novagen 1X binding buffer (5 mM imidazole,0.5 M NaCl, 20 mM Tris-HCl, pH 7.9)] cultures were prepared and used toaffinity purify soluble affinity-tagged protein as follows.

The pGBot lysate was affinity purified on a glutathione-agarose resin(Pharmacia) exactly as described in Smith and Corcoran [CurrentProtocols in Molecular Biology, Supplement 28 (1994), pp.16.7.1-16.7.7]. The pHisBot protein was purified on the His-Bind resin(Novagen) utilizing the His-bind buffer kit (Novagen) exactly asdescribed by manufacturer.

Samples from the purification of both the pGBot and pHisBot proteins(including uninduced, induced, total, soluble, and affinity-purifiedeluted protein) were resolved on SDS-PAGE gels. Followingelectrophoresis, proteins were analyzed by Coomassie staining or byWestern blot detection utilizing a chicken anti-C. botulinum Type Atoxoid antibody (as described in Example 22).

These studies showed that the pGBot protein was almost entirelyinsoluble under the utilized conditions, while the pHisBot protein wassoluble. Affinity purification of the pHisBot protein on this firstattempt was inefficient, both in terms of yield (most of theimmunoreactive botulinal protein did not bind to the His-bind resin) andpurity (the botulinal protein was estimated to comprise approximately20% of the total eluted protein).

d) Purification of Soluble C. botulinum C Fragment Protein SubstantiallyFree of Endotoxin Contamination

The above studies showed that the pHisBot protein was expressed in E.coli as a soluble protein. However, the affinity purification of thisprotein on the His-bind resin was very inefficient. In order to improvethe affinity purification of the soluble pHisBot protein (in terms ofboth yield and purity), an alternative poly-histidine binding affinityresin (Ni-NTA resin; Qiagen) was utilized. The Ni-NTA resin was reportedto have a superior binding affinity (Kd=1×10⁻¹³ at pH 8.0; Qiagen usermanual) relative to the His-bind resin.

A soluble lysate (in Novagen IX binding buffer) from an induced 1 liter2X YT culture was prepared as described above. Briefly, the culture ofpHisBot [Bl21(DE3)pLysS host] was grown at 37° C. to an OD₆₀₀ of 0.7 in1 liter of 2×YT medium containing 100 μg/ml ampicillin, 34 μg/mlchloramphenicol and 0.2% glucose. Protein expression was induced by theaddition of IPTG to 1 mM. Three hours after the addition of the IPTG,the cells were cooled for 15 min in an ice water bath and thencentrifuged 10 min at 5000 rpm in a JA10 rotor (Beckman) at 4° C. Thepellets were resuspended in a total volume of 40 mls Novagen 1X bindingbuffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9), transferredto two 35 ml Oakridge tubes and frozen at −70° C. for at least 1 hr. Thetubes were thawed and the cells were lysed by sonication (4×20 secondbursts using a Branson Sonifier 450 with a power setting of 6-7) on ice.The suspension was clarified by centrifugation for 20 min at 9,000 rpm(10,000×g) in a JA-17 rotor (Beckman).

The soluble lysate was brought to 0.1% NP40 and then was batch absorbedto 7 ml of a 1:1 slurry of Ni-NTA resin:binding buffer by stirring for 1hr at 4° C. The slurry was poured into a column having an internaldiameter of 1 or 2.5 cm (BioRad). The column was then washedsequentially with 15 mls of Novagen IX binding buffer containing 0.1%NP40, 15 ml of Novagen 1X binding buffer, 15 ml wash buffer (60 mMimidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9) and 15 ml NaHPO₄ washbuffer (50 mM NaHPO₄, pH 7.0, 0.3 M NaCl, 10% glycerol). The boundprotein was eluted by protonation of the resin using elution buffer (50mM NaHPO₄, pH 4.0, 0.3 M NaCl, 10% glycerol). The eluted protein wasstored at 4° C.

Samples of total, soluble and eluted protein were resolved by SDS-PAGE.Protein samples were prepared for electrophoresis as described inExample 22b. Duplicate gels were stained with Coomassie blue tovisualize the resolved proteins and C. botulinum type A toxin-reactiveprotein was detected by Western blot analysis as described in Example22b. A representative Coomassie stained gel is shown in FIG. 28. In FIG.28, the following samples were loaded on the 12.5% acrylamide gel. Lanes1-4 contain respectively total protein, soluble protein, soluble proteinpresent in the flow-through of the Ni-NTA column and affinity-purifiedpHisBot protein (i.e., protein released from the Ni-NTA resin byprotonation). Lane 5 contains high molecular weight protein markers(BioRad).

The purification of pHisBot protein resulted in a yield of 7 mg ofaffinity purified protein from a 1 liter starting culture ofBL21(DE3)pLysS cells harboring the pHisBot plasmid. The yield ofpurified pHisBot protein represented approximately 0.4% of the totalsoluble protein in the induced culture. Analysis of the purified pHisBotprotein by SDS-PAGE revealed that at least 90-95% of the protein waspresent as a single band (FIG. 28) of the predicted MW (50 kD). This 50kD protein band was immunoreactive with anti-C. botulinum type A toxinantibodies. The extinction coefficient of the protein preparation wasdetermined to be 1.4 (using the Pierce BCA assay) or 1.45 (using theLowry assay) OD₂₈₀ per 1 mg/ml solution.

Samples of pH neutralized eluted pHisBot protein were resolved on a KB803 HPLC column (Shodex). Although His-tagged proteins are retained bythis sizing column (perhaps due to the inherent metal binding ability ofthe proteins), the relative mobility of the pHisBot protein wasconsistent with that expected for a non-aggregated protein in solution.Most of the induced pHisBot protein was determined to be soluble underthe growth and solubilization conditions utilized above (i.e., greaterthan 90% of the pHisBot protein was found to be soluble as judged bycomparison of the levels of pHisBot protein seen in total and solubleprotein samples prepared from BL21(DE3)pLysS cells containing thepHisBot plasmid). SDS-PAGE analysis of samples obtained aftercentrifugation, extended storage at −20° C., and at least 2 cycles offreezing and thawing detected no protein loss (due to precipitation),indicating that the pHisBot protein is soluble in the elution buffer(i.e., 50 mM NaHPO₄, pH 4.0, 0.3 M NaCl, 10% glycerol).

Determination of endotoxin contamination in the affinity purifiedpHisBot preparation (after pH neutralization) using the LAL assay(Associates of Cape Cod) detected no significant endotoxincontamination. The assay was performed using the endpoint chromogenicmethod (without diazo-coupling) according to the manufacturer'sinstructions. This method can detect concentrations of endotoxin greaterthan or equal to 0.03 EU/ml (EU refers to endotoxin units). The LALassay was run using 0.5 ml of a solution comprising 0.5 mg pHisBotprotein in 50 mM NaHPO₄, pH 7.0, 0.3 M NaCl, 10% glycerol; 30-60 EU weredetected in the 0.5 ml sample. Therefore, the affinity purified pHisBotpreparation contains 60-120 EU/mg of protein. FDA Guidelines for theadministration of parenteral drugs require that a composition to beadministered to a human contain less than 5 EU/kg body weight (Theaverage human body weight is 70 kg; therefore up to 349 EU units can bedelivered in a parental dose). Because very small amount of protein areadministered in a vaccine preparation (generally in the range of 10-500μg of protein), administration of affinity purified pHisBot containing60-120 EU/mg protein would result in delivery of only a small percentageof the permissible endotoxin load. For example, administration of 10-500μg of purified pHisBot to a 70 kg human, where the protein preparationcontains 60 EU/mg protein, results in the introduction of only 0.6 to 30EU [i.e., 0.2 to 8.6% of the maximum allowable endotoxin burden perparenteral dose (less than 5 EU/kg body weight)].

The above results demonstrate that endotoxin (LPS) does not copurifywith the pHisBot protein using the above purification scheme.Preparations of recombinantly produced pHisBot protein containing lowerlevels of endotoxin (less than or equal to 2 EU/mg recombinant protein)may be produced by washing the Ni-NTA column with wash buffer until theOD₂₈₀ returns to baseline levels (i.e., until no more UV-absorbingmaterial comes off of the column).

The above results illustrate a method for the production andpurification of soluble, botulinal C fragment protein substantially freeof endotoxin.

Example 25 Optimization of the Expression and Purification of pHisBotProtein

The results shown in Example 24d demonstrated that the pHisBot proteinis an excellent candidate for use as a vaccine as it could be producedas a soluble protein in E. coli and could be purified free of pyrogenactivity. In order to optimize the, expression and purification of thepHisBot protein, a variety of growth and purification conditions weretested.

a) Growth Parameters

i) Host Strains

The influence of the host strain utilized upon the production of solublepHisBot protein was investigated. A large scale purification of pHisBotwas performed [as described in Example 24d above] using the BL21(DE3)host (Novagen) rather than the BL21(DE3)pLysS host. The deletion of thepLysS plasmid in the BL21(DE3) host yielded higher levels of expressiondue to de-repression of the plasmid's T7-lac promoter. However, theyield of affinity-purified soluble recombinant protein was very low(approximately 600 μg/liter culture) when purified under conditionsidentical to those described in Example 24d above. This result was dueto the fact that expression in the BL21(DE3) host yielded very highlevel expression of the pHisBot protein as insoluble inclusion bodies asshown by SDS-PAGE analysis of protein prepared from induced BL21(DE3)cultures (FIG. 29, lanes 1-7, described below). These resultsdemonstrate that the pHisBot protein is not inherently toxic to E. colicells and can be expressed to high levels using the appropriatepromoter/host combination.

FIG. 29 shows a Coomassie blue stained SDS-PAGE gel (12.5% acrylamide)onto which extracts prepared from BL21(DE3) cells containing the pHisBotplasmid were loaded. Each lane was loaded with 2.5 μl protein samplemixed with 2.5 μl of 2×SDS sample buffer. The samples were handled asdescribed in Example 22b. The following samples were applied to the gel.Lanes 1-7 contain protein isolated from the BL21(DE3) host. Lanes 8-14contain proteins isolated from the BL21(DE3)pLysS host. Total proteinwas loaded in lanes 1, 2, 4, 6, 8, 10 and 12. Soluble protein was loadedin Lanes 3, 5, 7, 9, 11 and 13. Lane 1 contains protein from uninducedhost cells. Lanes 2-13 contain protein from host cells induced for 3hours. IPTG was added to a final concentration of 0.1 mM (Lanes 6-7),0.3 mM (Lanes 4-5) or 1.0 mM (Lanes 2, 3, 8-13). The cultures were grownin LB broth (Lanes 8-9), 2X YT broth (Lanes 10-11) or terrific broth(Lanes 1-7, 12-13). The pHisBot protein seen in Lanes 3, 5 and 7 isinsoluble protein which spilled over from Lanes 2, 4 and 6,respectively. High molecular weight protein markers (BioRad) were loadedin Lane 14.

A variety of expression conditions were tested to determine if theBL21(DE3) host could be utilized to express soluble pHisBot protein atsuitably high levels (i.e, about 10 mg/ml). The conditions altered weretemperature (growth at 37 or 30° C.), culture medium (2X YT, LB orTerrific broth) and inducer levels (0.1, 0.3 or 1.0 mM IPTG). Allcombinations of these variables were tested and the induction levels andsolubility was then assessed by S9S-PAGE analysis of total and solubleextracts [prepared from 1 ml samples as described in Williams et al.,(1994), supra].

All cultures were grown in 15 ml tubes (Falcon #2057). All culturemedium was prewarmed overnight at the appropriate temperature and weresupplemented with 100 μg/ml ampicillin and 0.2% glucose. Terrific brothcontains 12 g/l bacto-tryptone, 24 g/l bacto-yeast extract and 100 ml/lof a solution comprising 0.17 M KH₂PO₄, 0.72 M K₂HPO₄. Cultures weregrown in an incubator on a rotating wheel (to ensure aeration) to anOD₆₀₀ of approximately 0.4, and induced by the addition of IPTG. In allcases, high level expression of insoluble pHisBot protein was observed,regardless of temperature, medium or inducer concentration.

The effect of varying the concentration of IPTG upon 2X YT culturesgrown at 23° C. was then investigated. IPTG was added to a finalconcentration of either 1 mM, 0.1 mM, 0.05 μM or 0.01 nM. At thistemperature, similar levels of pHisBot protein was induced in thepresence of either 1 or 0.1 mM IPTG; these levels of expression waslower than that observed at higher temperatures. Induced protein levelswere reduced at 0.05 mM IPTG and absent at 0.01 mM IPTG (relative to 1.0and 0.1 mM IPTG inductions at 23° C.). However, no conditions wereobserved in which the induced pHisBot protein was soluble in this host.Thus, although expression levels are superior in the BL21(DE3) host (ascompared to the BL21(DE3)pLysS host), conditions that facilitate theproduction of soluble protein in this host could not be identified.

These results demonstrate that production of soluble pHisBot protein wasachieved using the BL21(DE3)pLysS host in conjunction with the T7-lacpromoter.

ii) Effect of Varying Temperature, Medium and IPTG Concentration andLength of Induction

The effect growing the host cells in various mediums upon the expressionof recombinant botulinal protein from the pHisBot expression construct[in the BL21(DE3)pLysS host] was investigated. BL21(DE3)pLysS cellscontaining the pHisBot plasmid were grown in either LB, 2X YT orTerrific broth at 37° C. The cells were induced using 1 mM IPTG for a 3hr induction period. Expression of pHisBot protein was found to be thehighest when the cells were grown in 2X YT broth (see FIG. 29, lanes8-13).

The cells were then grown at 30° C. in 2X YT broth and the concentrationof IPTG was varied from 1.0, 0.3 or 0.1 mM and the length of inductionwas either 3 or 5 hours. Expression of pHisBot protein was similar atall 3 inducer concentrations utilized and the levels of induced proteinwere higher after a 5 hr induction as compared to a 3 hr induction.

Using the conditions found to be optimal for the expression of pHisBotprotein, a large scale culture was grown in order to provide sufficientmaterial for a large scale purification of the pHisBot protein. Three 1liter cultures were grown in 2X YT medium containing 100 μg/mlampicillin, 34 μg/ml chloramphenicol and 0.2% glucose. The cultures weregrown at 30° C. and were induced with 1.0 mM IPTG for a 5 hr period. Thecultures were harvested and a soluble lysate were prepared as describedin Example 18. A large scale purification was performed as described inExample 24d with the exception that except the soluble lysate was batchabsorbed for 3 hours rather than for 1 hour. The final yield was 13 mgpHisBot protein/liter culture. The pHisBot protein represented 0.75% ofthe total soluble protein.

The above results demonstrate growth conditions under which solublepHisBot protein is produced (i.e., use of the BL21(DE3)pLysS host, 2X YTmedium, 30° C., 1.0 mM IPTG for 5 hours).

b) Optimization of Purification Parameters

For optimization of purification conditions, large scale cultures (3×1liter) were grown at 30° C. and induced with 1 mM IPTG for 5 hours asdescribed above. The cultures were pooled, distributed to centrifugebottles, cooled and pelleted as described in Example 24d. The cellpellets were frozen at −70° C. until used. Each cell pellet represented⅓ of a liter starting culture and individual bottles were utilized foreach optimization experiment described below. This standardized theinput bacteria used for each experiment, such that the yields ofaffinity purified pHisBot protein could be compared between differentoptimization experiments.

i) Binding Specificity (pH Protonation)

A lysate of pHisBot culture was prepared in PBS (pH 8.0) and applied toa 3 ml Ni-NTA column equilibrated in PBS (pH 8.0) using a flow rate of0.2 ml/min (3-4 column volumes/hr) using an Econo chromatography system(BioRad). The column was washed with PBS (pH 8.0) until the absorbance(OD₂₈₀) of the elute was at baseline levels. The flow rate was thenincreased to 2 ml/min and the column was equilibrated in PBS (pH 7.0). ApH gradient (pH 7.0 to 4.0 in PBS) was applied in order to elute thebound pHisBot protein from the column. Fractions were collected andaliquots were resolved on SDS-PAGE gels. The PAGE gels were subjected toWestern blotting and the pHisBot protein was detected using a chickenanti-C. botulinum Type A toxoid antibody as described in Example 22.

From the Western blot analysis it was determined that the pHisBotprotein begins to elute from the Ni-NTA column at pH 6.0. This isconsistent with the predicted elution of a His-tagged protein monomer atpH 5.9.

These results demonstrate that the pH at which the pHisBot protein isprotonated (released) from Ni-NTA resin in PBS buffer is pH 6.0.

ii) Binding Specificity (Imidazole Competition)

In order to define purification conditions under which the native E.coli proteins could be removed from the Ni-NTA column while leaving thepHisBot protein bound to the column, the following experiment wasperformed. A lysate of pHisBot culture was prepared in 50 mM NaHPO₄, 0.5M NaCl, 8 mM imidazole (pH 7.0). This lysate was applied to a 3 mlNi-NTA column equilibrated in 50 mM NaHPO₄, 0.5 M NaCl (pH 7.0) using anEcono chromatography system (BioRad). A flow rate of 0.2 ml/min (3-4column volumes/hr) was utilized. The column was washed with 50 mMNaHPO₄, 0.5 M NaCl (pH 7.0) until the absorbance of the elute returnedto baseline. The flow rate was then increased to 2 ml/min.

The column was eluted using an imidazole step gradient [in 50 mM NaHPO₄,0.5 M NaCl (pH 7.0)]. Elution steps were 20 mM, 40 mM, 60 mM, 80 mM, 100mM, 200 mM, 1.0 M imidazole, followed by a wash using 0.1 mM EDTA (tostrip the nickel from the column and remove any remaining protein). Ineach step, the wash was continued until the OD₂₈₀ returned to baseline.Fractions were resolved on SDS-PAGE gels, Western blotted, and pHisBotprotein detected using a chicken anti-C. botulinum Type A toxoidantibody as described in Example 22. Duplicate gels were stained withCoomassie blue to detect eluted protein in each fraction.

The results of the PAGE analysis showed that most of thenon-specifically binding bacterial protein was removed by the 20 mMimidazole wash, with the remaining bacterial proteins being removed inthe 40 and 60 mM imidazole washes. The pHisBot protein began to elute at100 mM imidazole and was quantitatively eluted in 200 mM imidazole.

These results precisely defined the window of imidazole wash stringencythat optimally removes E. coli proteins from the column whilespecifically retaining the pHisBot protein in this buffer. These resultsprovided conditions under which the pHisBot protein can be purified freeof contaminating host proteins.

iii) Purification Buffers and Optimized Purification Protocols

A variety of purification parameters were tested during the developmentof an optimized protocol for batch purification of soluble pHisBotprotein. The results of these analyses are summarized below.

Batch purifications were performed (as described in Example 24d) usingseveral buffers to determine if alternative buffers could be utilizedfor binding of the pHisBot protein to the Ni-NTA column. It wasdetermined that quantitative binding of pHisBot protein to the Ni-NTAresin was achieved in either Tris-HCl (pH 7.9) or NaHPO₄ (pH 8.0)buffers. Binding of the pHisBot protein in NaHPO₄ buffer was notinhibited using 5 mM, 8 mM or 60 mM imidazole. Quantitative elution ofbound pHisBot protein was obtained in buffers containing 50 mM NaHPO₄,0.3 M NaCl (pH 3.5-4.0), with or without 10% glycerol. However,quantitation of soluble affinity purified pHisBot protein before andafter a freeze thaw (following several weeks storage of the affinitypurified elute at −20° C.) revealed that 94% of the protein wasrecovered using the glycerol-containing buffer, but only 68% of theprotein was recovered when the buffer lacking glycerol was employed.This demonstrates that glycerol enhanced the solubility of the pHisBotprotein in this low pH buffer when the eluted protein was stored atfreezing temperatures (e.g., −20° C.). Neutralization of pH by additionof NaH₂PO₄ buffer did not result in obvious protein precipitation.

It was determined that quantitative binding of pHisBot protein using thebatch format occurred after 3 hrs (FIG. 30), but not after 1 hr ofbinding at 4° C. (the resin was stirred during binding). FIG. 30 depictsa Coomassie blue stained SDS-PAGE gel (7.5% acrylamide) containingsamples of proteins isolated during the purification of pHisBot proteinfrom lysate prepared from the BL21(DE3)pLysS host. Each lane was loadedwith 5 μl of protein sample mixed with 5 μl of 2× sample buffer andprocessed as described in Example 22b. Lane 1 contains high molecularweight protein markers (BioRad). Lanes 2 and 3 contain protein elutedfrom the Ni-NTA resin. Lane 4 contains soluble protein after a 3 hrbatch incubation with the Ni-NTA resin. Lanes 5 and 6 contain solubleand total protein, respectively. FIG. 30 demonstrates that the pHisBotprotein is completely soluble [compare Lanes 5 and 6 which show that asimilar amount of the 50 kD pHisBot protein is seen in both; if asubstantial amount (greater than 20%) of the pHisBot protein werepartially insoluble in the host cell, more pHisBot protein would be seenin lane 6 (total protein) as compared to lane 5 (soluble protein)]. FIG.30 also demonstrates that the pHisBot protein is completely removed fromthe lysate after batch absorption with the Ni-NTA resin for 3 hours(compare Lanes 4 and 5).

The reported high affinity interaction of the Ni-NTA resin withHis-tagged proteins (K_(d)=1×10⁻¹³ at pH 8.0) suggested that it shouldbe possible to manipulate the resin-protein complexes withoutsignificant release of the bound protein. Indeed, it was determined thatafter the recombinant protein was bound to the Ni-NTA resin, theresin-pHisBot protein complex was highly stable and remained boundfollowing repeated rounds of centrifugation of the resin for 2 min at1600×g. When this centrifugation step was performed in a 50 ml tube(Falcon), a tight resin pellet formed. This allowed the removal of spentsoluble lysate by pouring off the supernatant followed by resuspensionof the pellet in wash buffer. Further washes can be performed bycentrifugation. The ability to perform additional washes permits thedevelopment of protocols for batch absorption of large volumes of lysatewith removal of the lysate being performed simply by centrifugationfollowing binding of the recombinant protein to the resin.

A simplified, integrated purification protocol was developed as follows.A soluble lysate was made by resuspending the induced cell pellet inbinding buffer [50 mM NaHPO₄, 0.5 M NaCl, 60 mM imidazole (pH 8.0)],sonicating 4×20 sec and centrifuging for 20 min at 10,000×g. NP-40 wasadded to 0.1% and Ni-NTA resin (equilibrated in binding buffer) wasadded. Eight milliliters of a 1:1 slurry (resin:binding buffer) was usedper liter of starting culture. The mixture was stirred for 3 hrs at 4°C. The slurry was poured into a column having a 1 cm internal diameter(BioRad), washed with binding buffer containing 0.1% NP40, then bindingbuffer until baseline was established (these steps may alternatively beperformed by centrifugation of the resin, resuspension in binding buffercontaining NP40 followed by centrifugation and resuspension in bindingbuffer). Imidazole was removed by washing the resin with 50 mM NaHPO₄,0.3M NaCl (pH 7.0). Protein bound to the resin was eluted using the samebuffer (50 mM NaHPO₄, 0.3M NaCl) having a reduced pH (pH 3.5-4.0).

A pilot purification was performed following this protocol and yielded18 mg/liter affinity-purified pHisBot. The pHisBot protein was greaterthan 90% pure as estimated by Coomassie staining of an SDS-PAGE gel.This represents the highest observed yield of soluble affinity-purifiedpHisBot protein and this protocol eliminates the need for separateimidazole-containing binding and wash buffers. In addition to providinga simplified and efficient protocol for the affinity purification ofrecombinant pHisBot protein, the above results provide a variety ofpurification conditions under which pHisBot protein can be isolated.

Example 26

The pHisBot Protein is an Effective Immunogen

In Example 23 it was demonstrated that neutralizing antibodies aregenerated in mouse serum after nasal immunization with the pMBotprotein. However, the pMBot protein was found to copurify withsignificant amounts of endotoxin which could not be easily removed. ThepHisBot protein, in contrast, could be isolated free of significantendotoxin contamination making pHisBot a superior candidate for vaccineproduction. To further assess the suitability of pHisBot as a vaccine,the immunogenicity of the pHisBot protein was determined and acomparison of the relative immunogenicity of pMBot and pHisBot proteinsin mice was performed as follows.

Two groups of eight BALBc mice were immunized with either pMBot proteinor pHisBot protein using Gerbu GMDP adjuvant (CC Biotech). pMBot protein(in PBS containing 10 mM maltose) or pHisBot protein (in 50 mMNaHPO₄,0.3 M NaCl, 10% glycerol, pH 4.0) was mixed with Gerbu adjuvant and usedto immunize mice. Each mouse received an IP injection of 100 μlantigen/adjuvant mix (50 μg antigen plus 1 μg adjuvant) on day 0. Micewere boosted as described above with the exception that the route ofadministration was IM on day 14 and 28. The mice were bled on day 77 andanti-C. botulinum Type A toxoid titers were determined using serumcollected from individual mice in each group (as described in Example23). The results are shown in Table 41. TABLE 41 Anti-C. botulinum TypeA Toxoid Serum IgG Titers In Individual Mice Immunized With pMBot orpHisBot Protein Preimmune¹ pMBot² pHisBot² Sample Dilution SampleDilution Sample Dilution Mouse # 1:50 1:250 1:1250 1:6250 1:50 1:2501:1250 1:6250 1:50 1:250 1:1250 1:6250 1 0.678 0.190 0.055 0.007 1.5740.799 0.320 0.093 2 1.161 0.931 0.254 0.075 1.513 0.829 0.409 0.134 31.364 0.458 0.195 0.041 1.596 1.028 0.453 0.122 4 1.622 1.189 0.3340.067 1.552 0.840 0.348 0.090 5 1.612 1.030 0.289 0.067 1.629 1.5800.895 0.233 6 0.913 0.242 0.069 0.013 1.485 0.952 0.477 0.145 7 0.9100.235 0.058 0.014 1.524 0.725 0.269 0.069 8 0.747 0.234 0.058 0.0141.274 0.427 0.116 0.029 Mean 0.048 0.021 0.011 0.002 1.133 0.564 0.1640.037 1.518 0.896 0.411 0.114 Titer¹The preimmune sample represents the average from 2 sets of duplicatewells containing serum from an individual mouse immunized withrecombinant Staphylococcus enterotoxin B (SEB) antigen. This antigen isimmunologically unrelated to C. Botulinum toxin and provides a controlserum.²Average of duplicate wells.

The results shown above in Table 41 demonstrate that both the pMBot andpHisBot proteins are immunogenic in mice as 100% of the mice (8/8) ineach group seroconverted from non-immune to immune status. The resultsalso show that the average titer of anti-C. botulinum Type A toxoid IgGis 2-3 fold higher after immunization with the pHisBot protein relativeto immunization with the pMBot protein. This suggests that the pHisBotprotein may be a superior immunogen to the pMBot protein.

Example 27

Immunization with the Recombinant pHisBot Protein Generates NeutralizingAntibodies

The results shown in Example 26 demonstrated that both the pHisBot andpMBot proteins were capable of inducing high titers of anti-C. botulinumtype A toxoid-reactive antibodies in immunized hosts. The ability of theimmune sera from mice immunized with either the pHisBot or pMBotproteins to neutralize C. botulinum type A toxoid in vivo was determinedusing the mouse neutralization assay described in Example 23b.

The two groups of eight BALBc mice immunized with either pMBot proteinor pHisBot protein in Example 26 were boosted again one week after thebleeding on day 77. The boost was performed by mixing pMBot protein (inPBS containing 10 mM maltose) or pHisBot protein (in 50 mM NaHPO₄, 0.3 MNaCl, 10% glycerol, pH 4.0) with Gerbu adjuvant as described in Example26. Each mouse received an IP injection of 100 μl antigen/adjuvant mix(50 μg antigen plus 1 μg adjuvant). The mice were bled 6 days after thisboost and the serum from mice within a group was pooled. Serum frompreimmune mice was also collected (this serum is the same serumdescribed in the footnote to Table 41).

The presence of neutralizing antibodies in the pooled or preimmune serumwas detected by challenging mice with 5 LD₅₀ units of type A toxin mixedwith 100 μl of pooled serum. The challenge was performed by mixing (permouse to be injected) 100 μl of serum from each pool with 100 μl ofpurified type A toxin standard (50 LD₅₀/ml prepared as described inExample 23b) and 500 μl of gel-phosphate. The mixtures were incubatedfor 30 min at room temperature with occasional mixing. Each of four micewere injected IP with the mixtures (0.7 ml/mouse). The mice wereobserved for signs of botulism for 72 hours. Mice receiving toxin mixedwith serum from mice immunized with either the pHisBot or pMBot proteinsshowed no signs of botulism intoxication. In contrast, mice receivingpreimmune serum died in less than 24 hours.

These results demonstrate that antibodies capable of neutralizing C.botulinum type A toxin are induced when either of the recombinant C.botulinum C fragment proteins pHisBot or pMBot are used as immunogens.

Example 28 Cloning and Expression of the C Fragment of C. botulinumSerotype A Toxin in E. coli Utilizing a Native Gene Fragment

In Example 22 above, a synthetic gene was used to express the C fragmentof C. botulinum serotype A toxin in E. coli. The synthetic gene replacednon-preferred (i.e., rare) codons present in the C fragment gene withcodons which are preferred by E. coli. The synthetic gene was generatedbecause it has been reported that genes which have a high A/T content(such as most clostridial genes) creates expression difficulties in E.coli and yeast. Furthermore, LaPenotiere et al. suggested that problemsencountered with the stability (non-fusion constructs) and solubility(MBP fusion constructs) of the C fragment of C. botulinum serotype Atoxin when expressed in E. coli was most likely due to the extreme A/Trichness of the native C. botulinum serotype A toxin gene sequences(LaPenotiere, et al, supra).

In this example, it was demonstrated that successful expression of the Cfragment of C. botulinum type A toxin gene in E. coli does not requirethe elimination of rare codons (i.e., there is no need to use asynthetic gene). This example involved a) the cloning of the native Cfragment of the C. botulinum serotype A toxin gene and construction ofan expression vector and b) a comparison of the expression andpurification yields of C. botulinum serotype A C fragments derived fromnative and synthetic expression vectors.

a) Cloning of the Native C Fragment of the C. botulinum Serotype A ToxinGene and Construction of an Expression Vector

The serotype A toxin gene was cloned from C. botulinum genomic DNA usingPCR amplification. The following primer pair was employed:5′-CGCCATGGCTAG ATTATTATCTACATTTAC-3′ (5′ primer, NcoI site underlined;SEQ ID NO:29) and 5′-GCAAGCTTCTTGACAGACTCATGTAG-3′ (3′ primer, HindIIIsite underlined; SEQ ID NO:30). C. botulinum type A strain was obtainedfrom the American Type Culture Collection (ATCC#19397) and grown underanaerobic conditions in Terrific broth medium. High molecular-weight C.botulinum DNA was isolated as described in Example 11. The integrity andyield of genomic DNA was assessed by comparison with a serial dilutionof uncut lambda DNA after electrophoresis on an agarose gel.

The gene fragment was cloned by PCR utilizing a proofreadingthermostable DNA polymerase (native Pfu polymerase). PCR amplificationwas performed using the above primer pair in a 50 μl reaction containing10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 200 μM each dNTP, 0.2μM each primer, and 50 ng C. botulinum genomic DNA. Reactions wereoverlaid with 100 μl mineral oil, heated to 94° C. 4 min, 0.5 μl nativePfu polymerase (Stratagene) was added, and thirty cycles comprising 94°C. for 1 min, 50° C. for 2 min, 72° C. for 2 min were carried outfollowed by 10 min at 72° C. An aliquot (10 μl) of the reaction mixturewas resolved on an agarose gel and the amplified native C fragment genewas gel purified using the Prep-A-Gene kit (BioRad) and ligated topCRScript vector DNA (Stratagene). Recombinant clones were isolated andconfirmed by restriction digestion, using standard recombinant molecularbiology techniques [Sambrook et al. (1989), supra]. In addition, thesequence of approximately 300 bases located at the 5′ end of the Cfragment coding region were obtained using standard DNA sequencingmethods. The sequence obtained was identical to that of the publishedsequence.

An expression vector containing the native C. botulinum serotype A Cfragment gene was created by ligation of the NcoI-HindIII fragmentcontaining the C fragment gene from the pCRScript clone to NheI-HindIIIrestricted pETHisa vector (Example 18b). The NcoI and NheI sites werefilled in using the Klenow enzyme prior to ligation; these sites werethus blunt-end ligated together. The resulting construct was termedpHisBotA (native). pHisBotA (native) expresses the C. botulinum serotypeA C fragment with a his-tagged N terminal extension which has thefollowing sequence:

MetGlyHisHisHisHisHisHisHisHisHisHisSerSerGlyHisIleGluGlyArgHisMetAla(SEQ ID NO:24), where the underlining represents amino acids encoded bythe C. botulinum C fragment gene (this N terminal extension contains therecognition site for FactorXa protease, shown in italics, which can beemployed to remove the polyhistidine tract from the N-terminus of thefusion protein). The pHisBot (native) construct expresses the identicalprotein as the pHisBot construct (Ex. 24c; herein after the pHisBotA)which contains the synthetic gene.

The predicted DNA sequence encoding the native C. botulinum serotype A Cfragment gene contained within pHisBotA (native) is listed in SEQ IDNO:3′ [the start of translation (ATG) is located at nucleotides 108-110and the stop of translation (TAA) is located at nucleotides 1494-1496 inSEQ ID NO:31] and the corresponding amino acid sequence is listed in SEQID NO:26 (i.e., the same amino acid sequence as that produced bypHisBotA containing synthetic gene sequences).

b) Comparison of the Expression and Purification Yields of C. botulinumSerotype A C Fragments Derived from Native and Synthetic ExpressionVectors

Recombinant plasmids containing either the native or the synthetic C.botulinum serotype A C fragment genes were transformed into E. colistrain Bl21(DE3) pLysS and protein expression was induced in 1 litershaker flask cultures. Total protein extracts were isolated, resolved onSDS-PAGE gels and C. botulinum C fragment protein was identified byWestern analysis utilizing a chicken anti-C. botulinum serotype A toxoidantiserum as described in Example 22.

Briefly, 1 liter (2XYT+100 μg/ml ampicillin and 34 μg/mlchloramphenicol) cultures of bacteria harboring either the pHisBotA(synthetic) or pHisBotA (native) plasmids in the Bl21(DE3) pLysS strainwere induced to express recombinant protein by addition of IPTG to 1 mM.Cultures were grown at 30-32° C., IPTG was added when the cell densityreached an OD₆₀₀ 0.5-1.0 and the induced protein was allowed toaccumulate for 3-4 hrs after induction.

The cells were cooled for 15 min in an ice water bath and thencentrifuged for 10 min at 5000 rpm in a JA10 rotor (Beckman) at 4° C.The cell pellets were resuspended in a total volume of 40 mls 1× bindingbuffer (40 mM imidazole, 0.5 M NaCl, 50 mM NaPO₄, pH 8.0), transferredto two 50 ml Oakridge tubes and frozen at −70° C. for at least 1 hr. Thetubes were then thawed and the cells were lysed by sonication (usingfour successive 20 second bursts) on ice. The suspension was clarifiedby centrifugation 20-30 min at 9,000 rpm (10,000 g) in a JA-17 rotor.The soluble lysate was batch absorbed to 7 ml of a 1:1 slurry of NiNTAresin:binding buffer by stirring 2-4 hr at 4° C. The slurry wascentrifuged for 1 min at 500 g in 50 ml tube (Falcon), resuspended in 5mls binding buffer and poured into a 2.5 cm diameter column (BioRad).The column was attached to a UV monitor (ISCO) and the column was washedwith binding buffer until a baseline was established Imidazole wasremoved by washing with 50 mM NaPO₄, 0.3 M NaCl, 10% glycerol, pH 7.0and bound protein was eluted using 50 mM NaPO₄, 0.3 M NaCl, 10%glycerol, pH 3.5-4.0.

The eluted proteins were stored at 4° C. Samples of total, soluble, andeluted proteins were resolved by SDS-PAGE. Protein samples were preparedfor electrophoresis by mixing 1 μl total (T) or soluble (S) protein with4 μl PBS and 5 μl 2×SDS-PAGE sample buffer, or 5 μl eluted (E) proteinand 5 μl 2×SDS-PAGE sample buffer. The samples were heated to 95° C. for5 min, then cooled and 5 or 10 μls were loaded on 12.5% SDS-PAGE gels.Broad range molecular weight protein markers (BioRad) were also loadedto allow the MW of the identified fusion proteins to be estimated. Afterelectrophoresis, protein was detected either generally by staining gelswith Coomassie blue, or specifically, by blotting to nitrocellulose forWestern blot detection of specific immunoreactive protein.

For Western blot analysis, the gels were blotted, and protein transferwas confirmed by Ponceau S staining as described in Example 22. Afterblocking the blots for 1 hr at room temperature in blocking buffer (PBSTand 5% milk), 10 ml of a 1/500 dilution of an anti-C. botulinum toxin AIgY PEG prep (Ex. 3) in blocking buffer was added and the blots wereincubated for an additional hour at room temperature. The blots werewashed and developed using a rabbit anti-chicken alkaline phosphataseconjugate (Boehringer ManNheIm) as the secondary antibody as describedin Ex. 22. This analysis detected C. botulinum toxin A-reactive proteinsin the pHisBotA (native and synthetic) protein samples (corresponding tothe predicted full length proteins identified by Coomassie staining).

A gel containing proteins expressed from the pHisBot and pHisBot(native) constructs during various stages of purification and stainedwith Coomassie blue is shown in FIG. 31. In FIG. 31, lanes 1-4 and 9contain proteins expressed by the pHisBotA construct (i.e., thesynthetic gene) and lanes 5-8 contain proteins expressed by the pHisBotA(native) construct. Lanes 1 and 5 contain total protein extracts; lanes2 and 6 contain soluble protein extracts; lanes 3 and 7 contain proteinswhich flowed through the NiNTA columns; lanes 4, 8 and 9 contain proteineluted from the NiNTA columns and lane 10 contains molecular weightmarkers.

The above purification resulted in a yield of 3 mg (native gene) or 11mg (synthetic gene) of affinity purified protein from a 1 liter startingculture, of which at least 90-95% of the protein was a single band ofthe predicted MW (50 kd) and immunoreactivity for recombinant C.botulinum serotype A C fragment protein. Other than the level ofexpression, no difference was observed between the native and thesynthetic gene expression systems.

These results demonstrate that soluble C. botulinum serotype A Cfragment protein can be expressed in E. coli and purified utilizingeither native or synthetic gene sequences.

Example 29 Generation of Neutralizing Antibodies Using a Recombinant C.botulinum Serotype A C Fragment Protein Containing a Six Residue His-Tag

In Example 27, neutralizing antibodies were generated utilizing thepHisBotA protein, which contains a histidine-tagged N-terminal extensioncomprising 10 histidine residues. To determine if the generation ofneutralizing antibodies is dependent on the presence of this particularhis-tag, a protein containing a shorter N-terminal extension (comprising6 histidine residues) was produced and tested for the ability togenerate neutralizing antibodies. This example involved a) the cloningand expression of the p6HisBotA(syn) protein and b) the generation andcharacterization of hyperimmune serum.

a) Cloning and Expression of the p6HisBotA(syn) Protein

The p6HisBotA(syn) construct was generated as described below; the term“syn” designates the presence of synthetic gene sequences. Thisconstruct expresses the C fragment of the C. botulinum serotype A toxinwith a histidine-tagged N terminal extension having the followingsequence: MetHisHisHisHisHisHisMetAla (SEQ ID NO:32); the amino acidsencoded by the botulinal C fragment gene are underlined and the vectorencoded amino acids are presented in plain type.

6×His oligonucleotides [5′-TATGCATCACCATCACCATCA-3′ (SEQ ID NO:33) and5′-CATGTGATGGTGATGGTGATGCA-3′ (SEQ ID NO:34) were annealed as follows.One microgram of each oligonucleotide was mixed in total of 20 μL 1×reaction buffer 2 (NEB) and the mixture was heated at 70° C. for 5 minand then incubated at 42° C. for 5 min. The annealed oligonucleotideswere then ligated with gel purified NdeI/HindIII cleaved pET23b (T7promoter) or pET21b (T7lac promoter) DNA and the gel purifiedNcoI/HindIII C. botulinum serotype A C fragment synthetic gene fragmentderived from pAlterBot (Ex. 22). Recombinant clones were isolated andconfirmed by restriction digestion. The DNA sequence encoding the6×his-tagged BotA protein contained within p6HisBotA(syn) is listed inSEQ ID NO:35. The amino acid sequence of the p6×HisBotA protein islisted in SEQ ID NO:36.

The resulting recombinant p6×HisBotA plasmid was transformed into theBL21(DE3)pLysS strain, and 1 liter cultures were grown, induced andharvested as described in Example 28. His-tagged protein was purified asdescribed in Example 28, with the following modifications. The bindingbuffer (BB) contained 5 mM imidazole rather than 40 mM imidazole andNP40 was added to the soluble lysate to a final concentration of 0.1%.The bound material was washed on the column with BB until the baselinewas established, then the column was washed successively with BB+20 μMimidazole and BB+40 mM imidazole. The column was eluted as described inExample 28.

In the case of the pET23-derived expression system, high levelexpression of insoluble 6HisBotA protein was induced. The pET21-derivedvector expressed lower levels of soluble protein that bound the NiNTAresin and eluted in the 40 mM imidazole wash rather than during the lowpH elution. These results (i.e., low level expression of a solubleprotein) are consistent with the results obtained with pHisBotA protein(Ex. 25); the pHisBotA construct, like the pET21-derived vector,contains the T7lac rather than T7 promoter.

The 6HisBotA protein thus elutes under less stringent conditions thanthe 10× histidine-containing pHisBot protein (100-200 mM imidazole; Ex.25) presumably due to the reduction in the length of the his-tag. Theeluted protein was of the predicted size [i.e., slightly reduced incomparison to pHisBotA protein].

b) Generation and Characterization of Hyperimmune Serum

Eight BALBc mice were immunized with purified 6HisBotA protein usingGerbu GMDP adjuvant (CC Biotech). The 40 mM imidazole elution was mixedwith Gerbu adjuvant and used to immunize mice. Each mouse received asubcutaneous injection of 100 μl antigen/adjuvant mix (12 μg antigen+1μg adjuvant) on day 0. Mice were subcutaneously boosted as above on day14 and bled on day 28. Control mice received pHisBotB protein (preparedas described in Ex. 35 below) in Gerbu adjuvant.

Anti-C. botulinum serotype A toxoid titers were determined in serum fromindividual mice from each group using the ELISA described in Example 23awith the exception that the initial testing serum dilution was 1:100 inblocking buffer containing 0.5% Tween 20, followed by serial 5-folddilutions into this buffer. The results of the ELISA demonstrated thatseroconversion (relative to control mice) occurred in all 8 mice.

The ability of the anti-C. botulinum serotype A C fragment antibodiespresent in serum from the immunized mice to neutralize native C.botulinum type A toxin was tested using the mouse neutralization assaydescribed in Example 23b. The amount of neutralizing antibodies presentin the serum of the immunized mice was determined using serum antibodytitrations. The various serum dilutions (0.01 ml) were mixed with 5 LD₅₀units of C. botulinum type A toxin and the mixtures were injected IPinto mice. The neutralizations were performed in duplicate. The micewere then observed for signs of botulism for 4 days. Undiluted serum wasfound to protect 100% of the injected mice while the 1:10 diluted serumdid not. This corresponds to a neutralization titer of 0.05-0.5 IU/ml.

These results demonstrate that neutralizing antibodies were induced whenthe 6HisBotA protein was utilized as the immunogen. Furthermore, theseresults demonstrate that seroconversion and the generation ofneutralizing antibodies does not depend on the specific N terminalextension present on the recombinant C. botulinum type A C fragmentproteins.

Example 30 Construction of Vectors for the Expression of His-Tagged C.botulinum Type A Toxin C Fragment Protein Using the Synthetic Gene

A number of expression vectors were constructed which contained thesynthetic C. botulinum type A toxin C fragment gene. These constructsvary as to the promoter (T7 or T7lac) and repressor elements (lacIq)present on the plasmid. The T7 promoter is a stronger promoter than isthe T7lac promoter. The various constructs provide varying expressionlevels and varying levels of plasmid stability. This example involved a)the construction of expression vectors containing the synthetic C.botulinum type A C fragment gene. and b) the determination of theexpression level achieved using plasmids containing either the kanamycinresistance or the ampicillin resistance genes in small scale cultures.

a) Construction of Expression Vectors Containing the Synthetic C.botulinum Type A C Fragment Gene

Expression vectors containing the synthetic C. botulinum type A Cfragment gene were engineered to utilize the kanamycin resistance ratherthan the ampicillin resistance gene. This was done for several reasonsincluding concerns regarding the presence of residual ampicillin inrecombinant protein derived from plasmids containing the ampicillinresistance gene. In addition, ampicillin resistant plasmids are moredifficult to maintain in culture; the β-lactamase secreted by cellscontaining ampicillin resistant plasmids rapidly degrades extracellularampicillin, allowing the growth of plasmid-negative cells.

A second altered feature of the expression vectors is the inclusion oflacIq gene in the plasmid. This repressor lowers expression from lacregulated promoters (the chromosomally located, lactose regulated T7polymerase gene and the plasmid located T7lac promoter). This downregulates uninduced protein expression and can enhance the stability ofrecombinant cell lines. The final alteration to the vectors is theinclusion of either the T7 or T7lac promoters that drive high ormoderate level expression of recombinant protein, respectively.

The expression plasmids were constructed as follows. In all cases, theprotein expressed is the pHisBotA(syn) protein previously described, andthe only differences between constructs is the alteration of the variousregulatory elements described above.

i) Construction of pHisBotA(syn) kan T7lac

The pHisBotA(syn) kan T7lac construct was made by inserting theSapI/XhoI fragment containing the C. botulinum type A C fragment frompHisBotA(syn) into pET24 digested with SapI/XhoI (Novagen; fragmentcontains kan gene and origin of replication). The desired construct wasselected for kanamycin resistance and confirmed by restrictiondigestion.

ii) Construction of pHisBotA(syn) kan lacIq T7lac

The pHisBotA(syn) kan lacIq T7lac construct was made by inserting theXbaI/HindIII fragment containing the C. botulinum type A C fragment frompHisBotA(syn)kanT7lac into the pET24a vector digested with XbaI/HindIII.The resulting construct was confirmed by restriction digestion.

iii) Construction of pHisBotA(syn) kan lacIq T7

The pHisBotA(syn) kan lacIq T7 construct was made by inserting theXbaI/HindIII fragment containing the C. botulinum type A C fragment frompHisBotA(syn) kan lacIq T7lac into XbaI/HindIII-digested pHisBotB(syn)kan lacIq T7 (described in Ex 37c below). The resulting construct wasconfirmed by restriction digestion.

b) Determination of the Expression Level Achieved Using PlasmidsContaining Either the Kanamycin Resistance or the Ampicillin ResistanceGenes in Small Scale Cultures

One liter cultures of pHisBotA(syn) kan T7lac/Bl21(DE3)pLysS andpHisBotA(syn) amp T7lac/Bl21(DE3)pLysS [this is the previouslydesignated pHisBotA(syn) construct] were grown, induced and his-taggedproteins were purified as described in Example 28. No differences inyield or protein integrity/purity were observed.

These results demonstrate that the antigen induction levels fromexpression constructs were not affected by the choice of ampicillinversus kanamycin antibiotic resistance genes.

Example 31 Fermentation of Cells Expressing Recombinant BotulinalProteins

a) Fermentation Culture of Cells Expressing Recombinant BotulinalProteins

Fermentation cultures were grown under the following conditions whichwere optimized for growth of the BL21(DE3) strains containing pETderived expression vectors. An overnight 1 liter feeder culture wasprepared by inoculating of 1 liter media (in a 2 L shaker flask) with afresh colony grown on an LB kan plate. The feeder culture contained: 600mls nitrogen source [20 gm yeast extract (BBL) and 40 gm tryptone(BBL)/600 mls], 200 mls 5× fermentation salts (per liter: 48.5 gmK₂HPO₄, 12 gm NaH₂PO₄H₂O, 5 gm NH₄Cl, 2.5 gm NaCl), 180 mls dH₂O, 20 mls20% glucose, 2 mls 1 M MgSO₄, 5 mls 0.05M CaCl₂ and 4 mls of a 10 mg/mlkanamycin stock. All solutions were sterilized by autoclaving, exceptthe kanamycin stock which was filter sterilized.

An aliquot (5 ml) of the feeder culture broth was removed prior toinoculation, and grown for 2 days at 37° C. as a culture broth sterilitycontrol. Growth was not observed in this control culture in any of thefermentations performed.

The inoculated feeder culture was grown for 12-15 hrs (ON) at 30-37° C.Care was taken to prevent oversaturation of this culture. The saturatedfeeder culture was added to 10 L of fermentation media in fermenter(BiofloIV, New Brunswick Scientific, Edison, N.J.) as follows. Thefermenter was sterilized 120 min at 121° C. with dH₂O. The sterile waterwas removed, and fermentation media added as follows: 6 liters nitrogensource, 2 liters 5× fermentation salts, 2 liters 2% glucose, 20 mls 1 MMgSO₄, 50 mls 0.05 M CaCl₂, 2.5-3.5 mls Macol P 400 antifoam (PPGIndustries Inc., Gurnee, Ill.), 40 mls 10 mg/ml kanamycin and 10 mlstrace elements (8 gm FeSO₄.7H₂O, 2 gm MnSO₄.H₂O, 2 gm AlCl₃.6H₂O, 0.8 gmCoCl.6H₂O, 0.4 gm ZnSO₄.7H₂O, 0.4 gm Na₂MoO₄.2H₂O, 0.2 gm CuCl₂.2H₂O,0.2 gm NiCl₂, 0.1 gm H₃BO₄/200 mls 5 M HCl). All solutions weresterilized by autoclaving, except the kanamycin stock which was filtersterilized. Fermentation media was prewarmed to 37° C. before theaddition of the feeder culture.

After the addition of the feeder culture, the culture was fermented at37° C., 400 rpm agitation, and 10 l/min air sparging. The DO₂ controlwas set to 20% PID and dissolved oxygen levels were controlled byincreasing the rate of agitation from 400-850 rpm under DO₂ control. DO₂levels were maintained at greater than or equal to 20% throughout theentire fermentation. When agitation levels reached 500-600 rpm thetemperature was lowered to 30° C. to reduce the oxygen consumption rate.Culture growth was continued until endogenous carbon sources weredepleted. In these fermentations, glucose was depleted first [monitoredwith a glucose monitoring kit (Sigma)], followed by assimilation ofacetate and other acidic carbons [monitored using an acetate test kit(Boehringer ManNheIm)]. During the assimilation phase, the pH rose from6.6-6.8 (starting pH) to 7.4-7.5, at which time the bulk of theremaining carbon source was depleted. This was signaled by a drop inagitation rate (from a maximum of 700-800 rpm) and a rise in DO₂levels>30%. This corresponds to a OD₆₀₀ reading of 18-20/ml. At thispoint a fed batch mode was initiated, in which a feed solution of 50%glucose was added at a rate of approximately 4 gm glucose/liter/hr. ThepH was adjusted to 7.0 by the addition of 25% H₃PO₄ (approximately 60mls). Culture growth was continued and reached peak oxygen consumptionwithin the next 3 hrs of growth (while the remaining residualnon-glucose carbon sources were assimilated). This phase ischaracterized by a slow increase in pH, and air sparging was increasedto 15 L/min, to keep the maximum rpm below 850.

Once the residual acidic carbon sources are depleted the agitation ratedecreases to 650-750 rpm and the pH begins to drop. pH control wasmaintained at 7.0 PID by regulated pump addition of a sterile 4M NaOHsolution which was consumed at a steady rate for the remainder of thefermentation. Growth was continued at 30° C., and the cultures weregrown linearly at a growth rate of 4-7 OD₆₀₀ units/hr, to at least 81.5OD₆₀₀ units/ml (>30 g/l dry cell weight) without induction. Antifoam (a1:1 dilution with filter sterilized 100% ethanol) was added as necessarythroughout the fermentation to prevent foaming.

During the fed batch mode, glucose was assimilated immediately(concentration in media consistently less than 0.1 gm/liter) and acetatewas not produced in significant levels by the pET plasmid/BL21(DE3) celllines tested (approximately 1 gm/liter at end of fermentation; this islower than that observed in harvests from shaker flask culturesutilizing the same strains). This was fortuitous, since high levels ofacetate has been shown to inhibit induction levels in a variety ofexpression systems. The above described conditions were found to behighly reproducible between fermentations and utilizing differentexpression plasmids. As a result, glucose and acetate level monitoringwere no longer preformed during fermentation.

b) Induction of Fermentation Cultures

Induction with IPTG (250 mg-10 gms, depending on the expression vectorand experiment) was initiated 1-3 hrs after initiation of the glucosefeed (30-50 OD₆₀₀/ml). The growth rate after induction was monitored ona hourly basis. Aliquots (5-10 ml) of cells were harvested at the timeof induction, and at hourly intervals post-induction. Optical densityreadings were determined by measuring the absorbance at 600 nm of 10 μlculture in 990 μl PBS versus a PBS control. The growth rate afterinduction was found to vary depending on the expression system utilized.

c) Monitoring of Fermentation Cultures

Fermentation cultures were monitored using the following control assays.

i) Colony Forming Ability

An aliquots of cells were removed from the cultures at each timepointsampled (uninduced and at various times after induction) were seriallydiluted in PBS (dilution 1=15 μl cells/3 ml PBS, dilution 2=15 μl ofdilution ⅓ ml PBS, dilution 3=3 or 6 μl of dilution ⅔ mls PBS) and 100μl of dilution 3 was plated on an LB or TSA (trypticase soy agar) plate.The plates were incubated ON at 37° C. and then the colonies are countedand scored for macro or micro growth.

ii) Phenotypic Characterization

Colonies growing on LB or TSA plates (above) from uninduced and inducedtimepoints were replica plated onto LB+kan, LB+chloramphenicol (forfermentations utilizing LysS or pACYCGro plasmids), LB+kan+1 mM IPTG andLB plates, in this order. The plates were grown 6-8 hrs at 37° C. andgrowth was scored on each plate for a minimum of 40-50 well isolatedcolonies. The percentage of cells retaining the plasmid at time ofinduction (i.e., uninduced cultures immediately prior to the addition ofIPTG) was determined to be the # colonies LB+Kan (or chlorarnphenicol)plate/# colonies LB plate×100%. The percentage of cells with mutated pETplasmids was determined to be the # colonies LB+Kan+IPTG plate/#colonies LB plate×100%. Colonies on all LB plates were scoredmorphologically for E. coli phenotype as a contamination control.Morphologically detectable contaminant colonies were not detected in anyfermentation.

iii) Recombinant BotA Protein Induction

A total of 10 OD₆₀₀0 units of cells (e.g., 200 μl of cells atOD₆₀₀=50/ml) were removed from each timepoint sample to a 1.5 mlmicrofuge tube and pelleted for 2 min at maximum rpm in a microfuge. Thepellets were resuspended in 1 ml of 50 mM NaHPO₄, 0.5 M NaCl, 40 mMimidazole buffer (pH 6.8) containing 1 mg/ml lysozyme. The samples wereincubated for 20 min at room temperature and stored ON at −70° C.Samples were thawed completely at room temperature and sonicated 2×10seconds with a Branson Sonifier 450 microtip probe at # 3 power setting.The samples were centrifuged for 5 min at maximum rpm in a microfuge.

An aliquot (20 μl) of the protein samples were removed to 20 μl 2×sample buffer, before or after centrifugation, for total and solubleprotein extracts, respectively. The samples were heated to 95° C. for 5min, then cooled and 5 or 10 μl were loaded onto 12.5% SDS-PAGE gels.High molecular weight protein markers (BioRad) were also loaded to allowfor estimation of the MW of identified fusion proteins. Afterelectrophoresis, protein was detected either generally by staining gelswith Coomassie blue, or specifically, by blotting onto nitrocellulose(as described in Ex. 28) for Western blot detection of specifichis-tagged proteins utilizing a NiNTA-alkaline phosphatase conjugateexactly as described by the manufacturer (Qiagen).

iv) Recombinant Antigen Purification

At the end of each fermentation run, 1-10 liters of culture wereharvested from the fermenter and the bacterial cells were pelleted bycentrifugation at 6000 rpm for 10 min in a JA10 rotor (Beckman). Thecell pellets were stored frozen at −70° C. or utilized immediatelywithout freezing. Cell pellets were resuspended to 15-20% weight tovolume in resuspension buffer (generally 50 mM NaPO₄, 0.5 M NaCl, 40 mMimidazole, pH 6.8) and lysed utilizing either sonication or highpressure homogenization.

For sonication, the resuspension buffer was supplemented with lysozymeto 1 mg/ml, and the suspension was incubated for 20 min. at room temp.The sample was then frozen ON at −70° C., thawed and sonicated 4×20seconds at microtip maximum to reduce viscosity. For homogenization, thecells were lyzed by 2 passes through a homogenizer (Rannie Mini-lab type8.30H) at 600 Bar. Cell lysates were clarified by centrifugation for 30min at 10,000 rpm in a JA10 rotor.

For IDA chromatography, samples were flocculated utilizingpolyethyleneimine (PEI) prior to centrifugation. Cell pellets wereresuspended in cell resuspension buffer (CRB: 50 mM NaPO₄, 0.5 M NaCl,40 mM imidazole, pH 6.8) to create a 20% cell suspension (wet weight ofcells/volume of CRB) and cell lysates were prepared as described above(sonication or homogenization). PEI (a 2% solution in dH₂O, pH 7.5 withHCl) was added to the cell lysate a final concentration of 0.2%, andstirred for 20 min at room temperature prior to centrifugation (8,500rpm in JA10 rotor for 30 minutes at 4° C.). This treatment removed RNA,DNA and cell wall components, resulting in a clarified, low viscositylysate (“PEI clarified lysate”).

His-tagged proteins were purified from soluble lysates by metal-chelateaffinity chromatography using either a NiNTA resin (as described in Ex.28) or an IDA (iminodiacetic acid) resin as described below.

IDA resin affinity purifications were performed utilizing a low pressurechromatography system (ISCO). A 7 ml (small scale) or 70 ml (largescale) Chelating Sepharose Fast Flow (Pharmacia) affinity column waspoured; in addition, a second guard column was poured and attached inline with the first column (to capture Ni ions that leached off theaffinity column). The columns were washed with 3 column volumes of dH₂O.The guard column was then removed and the affinity column was washedwith 0.3 M NiSO₄ until resistivity was established, then with dH₂O untilthe resistivity returned to baseline. The columns were reconnected andequilibrated with cell resuspension buffer (CRB; 50 mM NaPO₄, 0.5 MNaCl, 40 mM imidazole, pH 6.8). The clarified sample (in CRB) wasloaded. Flow rates were 5 ml/min for small scale columns and 20 m/minfor large scale columns. After sample loading, the column was washedwith CRB until a baseline established and bound protein was eluted withelution buffer (50 mM NaPO₄, 0.5 M NaCl, 800 mM imidazole, 20% glycerol,pH 6.8 or 8.0). Protein samples were stored at 4° C. or −20° C. Theyield of eluted protein was established by measuring the OD₂₈₀ of theelutions, with a 1 mg/ml solution of protein assumed to yield anabsorbance reading of 2.0.

The IDA columns may be regenerated and reused multiple times (>10). Toregenerate the column, the column was washed with 2-3 column volumes ofH₂O, then 0.05 M EDTA until all of the blue/green color was removedfollowed by a wash with dH₂O. The IDA columns were sterilized with 0.1 MNaOH (using at least 3 column volumes but not more than 50 minutescontact time with column packing material), then washed with 3 columnvolumes 0.05 M NaPO₄, pH 5.0, then dH₂O and stored at room temperaturein 20% ethanol.

Example 32 Construction of a Folding Chaperone Overexpression System

Co-overexpression of the E. coli GroEL/GroES folding chaperones in acell expressing a recombinant foreign protein has been reported toenhance the solubility of some foreign proteins that are otherwiseinsoluble when expressed in E. coli [Gragerouu et al. (1992) Proc. Natl.Acad. Sci. USA 89:10344]. The improvement in solubility is thought to bedue to chaperone-mediated binding and unfolding of insoluble denaturedproteins, thus allowing multiple attempts for productive refolding ofrecombinant proteins. By overexpressing the chaperones, theunfolding/refolding reaction is driven by excess chaperone, resulting,in some cases, in higher yields of soluble protein.

In this example, a chaperone overexpression system, compatible with pETvector expression systems, was constructed to facilitate testingchaperone-mediated solubilization of C. botulinum type A proteins. Thisexample involved the cloning of the GroEL/ES operon and construction ofa pLysS-based chaperone hyperexpression system.

The GroEL/GroES operon was PCR amplified and cloned into the pCRScriptvector as described in Example 28. The following primer pair was used:5′-CGCAT ATGAATATTCGTCCATTGCATG-3′ (SEQ ID NO:37) [5′ primer, startcodon of groES gene converted to NdeI site (underlined)] and5′-GGAAGCTTGCAGGGCAAT TACATCATG (SEQ ID NO:38) (3′ primer, stop codon ofgroEL gene italicized, engineered HindIII site underlined).Following-amplification, the chaperone operon was excised as anNdeI/HindIII fragment and cloned into pET23b digested with NdeI andHindIII. This construction places the Gro operon under the control ofthe T7 promoter of the pET23 vector. The desired construct was confirmedby restriction digestion.

The T7 promoter-Gro operon-T7 terminator expression cassette was thenexcised as a BglII/BSpEI (filled) fragment and cloned into BamHI(compatible with BglII)/HindIII (filled) cleaved pLysS plasmid (thisremoved the T7 lysozyme gene). The resulting construct was designatedpACYCGro, since the plasmid utilizing the pACYC184 origin from the plysSplasmid. Proper construction was confirmed by restriction digestion.

pACYCGro was transformed into BL21(DE3), cultures were grown and inducedwith 1 mM IPTG as described in preceding examples. Total and solubleprotein extracts were generated from cells removed before and after IPTGinduction and were resolved on a 12.5% SDS-PAGE gel and stained withCoomassie blue. This analysis revealed that high levels of soluble GroEIand GroES proteins were made in the induced cells. These resultsdemonstrated that the chaperone hyper-expression system was functional.

Example 33 Growth of BotA/pACYCGro Cell Lines in Fermentation Cultures

Induction of BL21(DE3) cells lacking the LysS plasmid which containedBotA expression constructs grown in shaker flask or fermentation cultureresulted in the expression of primarily insoluble BotA protein.Fermentation cultures were performed to determine if the simultaneousoverexpression of the Gro operon and recombinant C. botulinum type Aproteins (BotA proteins) resulted in enhanced solubility of therecombinant BotA protein. This example involved the fermentation ofpHisBotA(syn)kan lacIq T7lac/pACYCGro BL21(DE3) and pHisBotA(syn)kanlacIq T7/pACYCGro BL21(DE3) cell lines. The fermentations were repeatedexactly as described in Example 31. Chloramphenicol (34 μg/ml) wasincluded in the feeder and fermentation cultures.

a) Fermentation of pHisBotA(syn)kan lacIq T7lac/pACYCGro BL21(DE3) Cells

For fermentation of cells containing plasmids comprising the T7lacpromoter, induction was with 2 gms IPTG at 1 hr post initiation ofglucose feed. The OD₆₀₀ was 35 at time of induction, then 48.5, 61.5, 67at 1-3 hrs post induction. Viable colony counts decreased from 0-3 hrinduction [21 (13), 0, 0, 0; dilution 3 utilized 3 μl of dilution 2cells] with numbers in parenthesis for the indicating microcolonies. Of28 colonies scored at the time of induction, 23 retained thepHisBotA(syn)kan lacIq T7lac plasmid (kan resistant), 22 contained thechaperone plasmid (chloramphenicol resistant) and no colonies atinduction grew on IPTG+Kan plates (no mutations detected). These resultswere indicative of very strong promoter induction, since colonyviability dropped immediately after induction.

Total and soluble extracts were resolved on a 12.5% SDS-PAGE gel andstained with Coomassie. High level induction of Gro chaperones wasobserved, but very low level expression of soluble BotA protein wasobserved, increasing from 1 to 4.0 hrs post induction (no expressiondetected in uninduced cells). The dramatically lower expression of theBotA antigen in the presence of chaperone may be due to promoterocclusion (i.e., the stronger T7 promoter on the chaperone plasmid ispreferentially utilized).

b) Fermentation of pHisBotA(syn)kan lacIq T7/pACYCGro BL21(DE3) Cells

A fermentation utilizing the T7-driven BotA expression plasmid wasperformed. Induction was with 1 gm IPTG at 2 hrs post initiation ofglucose feed. The OD₆₀₀ was 41 at time of induction, then 51.5, 61.5,61.5 and 66 at 1-4 hrs post induction. Viable colony counts decreasedfrom 0-4 hrs induction [71, 1 (34), 1 (1), 1, 0; dilution 3 utilized 6μl dilution 2 cells) with numbers in parenthesis for the uninducedtimepoint indicating microcolonies. Of 65 colonies scored at the time ofinduction, all 65 retained both the pHisBotA(syn)kan lacIq T7 plasmid(kan resistant) and the chaperone plasmid (chloramphenicol resistant)and no colonies at induction grew on IPTG+Kan plates (no mutationsdetected).

Total and soluble extracts were resolved on a 12.5% SDS-PAGE gel andstained with Coomassie. High level induction of Gro chaperones andmoderate level expression of soluble BotA protein was observed,increasing from 1 to 4.0 hrs post induction (no expression detected inuninduced cells).

A PEI-clarified lysate (0.2% final concentration PEI) [850 ml from 130gm cell pellet (2 liters fermentation harvest)] was purified on a largescale IDA column. A total of 78 mg of protein was eluted. Extracts fromthe purification were resolved on a 12.5% SDS-PAGE gel and stained withCoomassie. The elution was found to contain an approximately 1:1 mix ofBotA/chaperone protein (FIG. 32). PEI lysates prepared in this mannerwere typically 16 OD₂₈₀/ml. This was estimated to be 8 mg protein/ml oflysate (by BCA assay). Thus, the eluted recombinant BotA proteinrepresented 0.55% of the total soluble cellular protein applied to thecolumn.

In FIG. 32, lane 1 contains molecular weight markers, lanes 2-9 containextracts from pHisBotA(syn)kan lacIq T7/pACYCGro/BL21(DE3) cells beforeor during purification on the IDA column. Lane 2 contains total proteinextract; lane 3 contains soluble protein extract; lanes 4 and 5 containPEI-clarified lysates (duplicates); lanes 6 and 7 contain flow-throughfrom the IDA column (duplicates) and lanes 8 and 9 contain IDA columnelute (lane 9 contains 1/10 the amount applied to lane 8).

These results demonstrate, that although the majority of the BotAprotein produced was insoluble, 20 mg/liter of soluble recombinant BotAprotein can be purified utilizing the pHisBotA(syn)kan lacIqT7/pACYCGro/BL21(DE3) expression system.

Example 34 Purification of Recombinant BotA Protein from FoldingChaperones

In this example of size exclusion chromatography was used to purify therecombinant BotA protein away from the folding chaperones and imidazolepresent in the IDA-purified material (Ex. 33).

To enhance the solubility of the recombinant BotA protein duringscale-up, the protein was co-expressed with folding chaperones (Ex. 33).As observed with the recombinant BotB protein (Example 40 below), thefolding chaperones co-eluted with the recombinant BotA protein duringthe Ni-IDA purification step. Because the recombinant BotA and BotBproteins have similar molecular weights (about 1/10 the size of thenon-reduced folding chaperone) and the imidazole step gradient strategywas unsuccessful in purifying BotB away from the folding chaperone (seeEx. 40), size exclusion chromatography was examined for the ability topurify the recombinant BotA protein away from the folding chaperones.

A column (2.5×24 cm) containing Sephacryl S-100 HR (Pharmacia) waspoured (bed volume ˜110 ml). Proteins having molecular weights greaterthan 100 K are expected to elute in the void volume under theseconditions and smaller proteins should be retained by the beads andelute at different times, depending on their molecular weights. Tomaintain solubility of the purified BotA protein, the Sephacryl columnwas equilibrated in a buffer having the same salt concentration as thebuffer used to elute the BotA protein from the IDA column (i.e., 50 mMsodium phosphate, 0.5 M NaCl, 10% glycerol; all reagents fromMallinkrodt, Chesterfield, Mo.).

Five milliliters of the IDA-purified recombinant BotA protein (Ex. 33)was filtered through a 0.45μ syringe filter, applied to the column andthe equilibration buffer was pumped through the column at a flow rate of1 ml/minute. Eluted proteins were monitored by absorbance at 280 nm andcollected either manually or with a fraction collector (BioRad).Appropriate fractions were pooled, if necessary, and the protein wasquantitated by absorbance at 280 nm and/or BCA protein assay (Pierce).The isolated peaks were then analyzed by native and/or SDS-PAGE toidentify the proteins present and to evaluate purity. The foldingchaperone eluted first, followed by the recombinant BotA protein andthen the imidazole peak.

SDS-PAGE analysis (12.5% polyacrylamide, reduced samples) was used toevaluate the purity of the IDA-purified recombinant BotA protein beforeand after S-100 purification. FIG. 33 shows the difference in puritybefore and after the S-100 purification step. In FIG. 33, lane 1contains molecular weight markers (BioRad broad range). Lane 2 shows theIDA-purified recombinant BotA protein preparation, which is contaminatedwith significant amounts of the folding chaperone. Following S-100purification, the amount of folding chaperone present in the BotA sampleis reduced dramatically (lane 3). Lane 4 contains no protein (i.e., itis a blank lane); lanes 5-8 contain samples of IDA-purified recombinantBotB and BotE proteins and are discussed infra.

Endotoxin levels in the S-100 purified BotA preparation were determinedusing the LAL assay (Associates of Cape Cod) as describe in Example 24.The purified BotA preparation was found to contain 22.7 to 45.5 EU/mgrecombinant protein.

These results demonstrate that size exclusion chromatography wassuccessful in purifying the recombinant BotA protein from foldingchaperones and imidazole following an initial IDA purification step.Furthermore, these results demonstrate that the S-100 purified BotAprotein was substantially free of endotoxin.

Example 35 Cloning and Expression of the C Fragment of the C. botulinumSerotype B Toxin Gene

The C. botulinum type B neurotoxin gene has been cloned and sequenced[Whelan et al. (1992) Appl. Environ. Microbiol. 58:2345 and Hutson etal. (1994) Curr. Microbiol. 28:101]. The nucleotide sequence of thetoxin gene derived from the Eklund 17B strain (ATCC 25765) is availablefrom the EMBL/GenBank sequence data banks under the accession numberX71343; the nucleotide sequence of the coding region is listed in SEQ.ID NO:39. The amino acid sequence of the C. botulinum type B neurotoxinderived from the strain Eklund 17B is listed in SEQ ID NO:40. Thenucleotide sequence of the C. botulinum serotype B toxin gene derivedfrom the Danish strain is listed in SEQ ID NO:41 and the correspondingamino acid sequence is listed in SEQ ID NO:42.

The DNA sequence encoding the native C. botulinum serotype B C fragmentgene derived from the Eklund 17B strain can be expressed using thepETHisb vector; the resulting coding region is listed in SEQ ID NO:43and the corresponding amino acid sequence is listed in SEQ ID NO:44. TheDNA sequence encoding the native C. botulinum serotype B C fragment genederived from the Danish strain can be expressed using the pETHisbvector; the resulting coding region is listed in SEQ ID NO:45 and thecorresponding amino acid sequence is listed in SEQ ID NO:46. The Cfragment region from any strain of C. botulinum serotype B can beamplified and expressed using the approach illustrated below using the Cfragment derived from C. botulinum type B 2017 strain.

The C. botulinum type B neurotoxin gene is synthesized as a singlepolypeptide chain which is processed to form a dimer composed of a lightand a heavy chain linked via disulfide bonds; the type B neurotoxin hasbeen reported to exist as a mixture of predominantly single chain withsome double chain (Whelan et al., supra). The 50 kD carboxy-terminalportion of the heavy chain is referred to as the C fragment or the H_(C)domain. Expression of the C fragment of C. botulinum type B toxin inheterologous hosts (e.g., E. coli) has not been previously reported.

The native C fragment of the C. botulinum serotype B toxin gene wascloned and expression constructs were made to facilitate proteinexpression in E. coli. This example involved PCR amplification of thegene, cloning, and construction of expression vectors.

The C fragment of the C. botulinum serotype B (BotB) toxin gene wascloned using the protocols and conditions described in Example 28 forthe isolation of the native BotA gene. The C. botulinum type B 2017strain was obtained from the American Type Culture Collection (ATCC#17843). The following primer pair was used to amplify the BotB gene:5′-CGCCATGGCTGATACAATACTAATAGAA ATG-3′ [5′ primer, engineered NcoI siteunderlined (SEQ ID NO:47)] and 5′-GCAAGCTTTATTCAGTCCACCCTTCATC-3′ [3′primer, engineered HindIII site underlined, native gene terminationcodon italicized (SEQ ID NO:48)]. After cloning into the pCRscriptvector, the NheI(filled)/HindIII fragment was cloned into pETHisb vectoras described for BotA C fragment gene in Example 28. The resultingconstruct was termed pHisBotB. pHisBotB expresses the BotB genesequences under the transcriptional control of the T7 lac promoter andthe resulting protein contains an N-terminal 10×His-tag affinity tag.The pHisBotB expression construct was transformed into BL21 (DE3) pLysScompetent cells and 1 liter cultures were grown, induced and his-taggedproteins were purified utilizing a NiNTA resin (eluted in low pH elutionbuffer) as described in Example 28. Total, soluble and purified proteinswere resolved by SDS-PAGE and detected by Coomassie staining and Westernblot hybridization utilizing a chicken anti-C. botulinum serotype Btoxoid primary antibody (generated by immunization of hens using C.botulinum serotype B toxoid as described in Example 3). Samples of BotAand BotE C fragment proteins were included on the gels for MW andimmunogenicity comparisons. Strong immunoreactivity to only the BotBprotein was detected with the anti-C. botulinum serotype B toxoidantibodies. The recombinant BotB protein was expressed at low levels (3mg/liter) as a soluble protein. The purified BotB protein migrated as asingle band of the predicted MW (i.e., ˜50 kD).

These results demonstrate the cloning of the native C. botulinumserotype B C fragment gene, the expression and purification of therecombinant BotB protein as a soluble his-tagged protein in E. coli.

Example 36 Generation of Neutralizing Antibodies Using the RecombinantpHisBotB Protein

The ability of the purified pHisBot protein to generate neutralizingantibodies was examined. Nine BALBc mice were immunized with BotBprotein (purified as described in Ex. 35) using Gerbu GMDP adjuvant (CCBiotech). The low pH elution was mixed with Gerbu adjuvant and used toimmunize mice. Each mouse received a subcutaneous injection of 100 μlantigen/adjuvant mix (15 μg antigen+1 μg adjuvant) on day 0. Mice weresubcutaneously boosted as above on day 14 and bled on day 28. Mice weresubsequently boosted 1-2 weeks after bleeding and were then bled on day70.

Anti-C. botulinum serotype B toxoid titers were determined in day 28serum from individual mice from each group using the ELISA protocoloutlined in Example 29 with the exception that the plates were coatedwith C. botulinum serotype B toxoid, and the primary antibody was achicken anti-C. botulinum serotype B toxoid. Seroconversion [relative tocontrol mice immunized with pHisBotE antigen (described below)] wasobserved with all 9 mice immunized with the purified pHisBotB protein.

The ability of the anti-BotB antibodies to neutralize native C.botulinum type B toxin was tested in a mouse-C. botulinum neutralizationmodel using pooled mouse serum (see Ex. 23b). The LD₅₀ of purified C.botulinum type B toxin complex (Dr. Eric Johnson, University ofWisconsin, Madison) was determined by a intraperitoneal (IP) method[Schantz and Kautler (1978), supra] using 18-22 g female ICR mice. Theamount of neutralizing antibodies present in the serum of the immunizedmice was determined using serum antibody titrations. The various serumdilutions (0.01 ml) were mixed with 5 LD₅₀ units of C. botulinum type Btoxin and the mixtures were injected IP into mice. The neutralizationswere performed in duplicate. The mice were then observed for signs ofbotulism for 4 days. Undiluted serum (day 28 or day 70) was found toprotect 100% of the injected mice while the 1:10 diluted serum did not.This corresponds to a neutralization titer of 0.05-0.5 IU/ml.

These results demonstrate that seroconversion occurred and neutralizingantibodies were induced when the pHisBotB protein was utilized as theimmunogen.

Example 37 Construction of Vectors to Facilitate Expression ofHis-Tagged BotB Protein in Fermentation Cultures

A number of expression vectors were constructed to facilitate theexpression of recombinant BotB protein in large scale fermentationculture. These constructs varied as to the strength of the promoterutilized (T7 or T7lac) and the presence of repressor elements (lacIq) onthe plasmid. The resulting constructs varied in the level of expressionachieved and in plasmid stability which facilitated the selection of anoptimal expression system for fermentation scaleup.

The BotB expression vectors created for fermentation culture wereengineered to utilize the kanamycin rather than the ampicillinresistance gene, and contained either the T7 or T7lac promoter, with orwithout the lacIq gene for the reasons outlined in Example 30.

In all cases, the protein expressed by the various expression vectors isthe pHisBot B protein described in Example 35, with the only differencesbetween clones being the alteration of various regulatory elements.Using the designations outlined below, the pHisBotB clone (Ex. 35) isequivalent to pHisBotB amp T7lac.

a) Construction of pHisBotB kan T7lac

pHisBotB kan T7lac was constructed by insertion of the BglII/HindIIIfragment of pHisBotB which contains the BotB gene sequences into thepPA1870-2680 kan T7lac vector which had been digested with BglII andHindIII (the pPA1870-2680 kan T7lac vector contains the pET24 kan genein the pET23 vector, such that no lacIq gene is present). Properconstruction of pHisBotB kan T7lac was confirmed by restrictiondigestion.

b) Construction of pHisBotB kan lacIq T7lac

pHisBotB kan lacIq T7lac was constructed by insertion of theBglII/HindIII fragment of pHisBotB which contains the BotB genesequences into similarly cut pET24a vector. Proper construction ofpHisBotB kan lacIq T7lac was confirmed by restriction digestion.

c) Construction of pHisBotB kan lacIq T7

pHisBotB kan lacIq T7 was constructed by inserting the NdeI/XhoIfragment from pHisBotE kan lacIq T7lac which contains the BotB genesequences into similarly cleaved pPA1870-2680 kan lacIq T7 vector (thisvector contains the T7 promoter, the same N-terminal his-tag as the Botconstructs, the C. difficile toxin A insert, and the kan lacIq genes;this cloning replaces the C. difficile toxin A insert with the BotBinsert). Proper construction was confirmed by restriction digestion.

Expression of recombinant BotB protein from these expression vectors andpurification of the BotB protein is described in Example 38 below.

Example 38 Fermentation and Purification of Recombinant BotB ProteinUtilizing the pHisBotB kan lacIq T7lac, pHisBotB kan T7lac and pHisBotBkan lacIq T7 Vectors

The pHisBotB kan lacIq T7lac, pHisBotB kan T7lac and BotB kan lacIq T7constructs [all transformed into the Bl21(DE3) strain] were grown infermentation cultures to determine the utility of the various constructsfor large scale expression and purification of soluble BotB protein. Allfermentations were performed as described in Example 31.

a) Fermentation of pHisBotB kan lacIq T7lac/Bl21(DE3) Cells

The fermentation culture was induced 45 min post start of glucose feedwith 1 gm IPTG (final concentration=0.4 mM). pH was maintained at 6.5rather than 7.0. The OD₆₀₀ was 27 at time of induction, then 35, 38, and40 at 1-3 hrs post induction. Duplicate platings of diluted 1 hrinduction samples (dilutions were prepared as described Ex. 31, dilution3 utilized 3 μl of dilution 2 cells) on TSA and LB+kan plates yielded 89TSA colonies and 81 kan colonies (90% kan resistant).

Total and soluble protein extracts were resolved on a 12.5% SDS-PAGE geland total protein was detected by staining with Coomassie blue. Lowlevel induction of insoluble Bot B protein was observed, increasing from1 to 3 hrs post induction (no expression was detected in uninducedcells).

b) Fermentation of pHisBotB kan T7lac/Bl21(DE3) Cells

The fermentation culture was induced 1 hr post start of glucose feedwith 2 gm IPTG (final concentration=0.8 mM). pH was maintained at 6.5rather than 7.0. The OD₆₀₀ was 24.5 at time of induction, then 31.5, 32,and 33 at 1-3 hrs post induction, respectively. Duplicate platings ofdiluted 0 hr and 2 hr induction samples (dilutions were prepared asdescribed Ex. 31; dilution 3 utilized 3 μl of dilution 2 cells) on TSAand LB+kan plates yielded 32 TSA colonies and 54 kan colonies (all kanresistant) for uninduced cells, and 1 TSA colony and 0 kan colonies 2 hrpost induction. These results were indicative of strong induction, sinceviable counts decreased dramatically 2 hrs post induction.

Total and soluble extracts were resolved on a 10% SDS-PAGE gel and totalprotein was detected by staining with Coomassie blue. Moderate inductionof insoluble BotB protein was observed, increasing from 1 to 3 hrs postinduction (no expression was detected in uninduced cells).

c) Fermentation of pHisBotB kan lacIq T7/Bl21(DE3) Cells

The fermentation was induced 2 hr post start of glucose feed with 4 gmIPTG (final concentration=1.6 mM). pH was maintained at 6.5 rather than7.0. The OD₆₀₀ was 45 at time of induction, then 47, 50, and 50 and 55at 1-4 hrs post induction, respectively. Viable colony counts decreasedafter induction (96, 1, 1, 2, 3; dilution 3 utilized 3 μl of dilution 2cells). Of 63 colonies scored at the time of induction, all 63 retainingthe BotB plasmid (kan resistant) and no colonies at induction grew onIPTG+Kan plates (no mutations detected).

Total and soluble extracts were resolved on a 12.5% SDS-PAGE gel andtotal protein was detected by staining with Coomassie blue. Moderatelevel induction of insoluble BotB protein was observed, increasing from1 to 4 hrs post induction (lower level expression was detected inuninduced cells, since the T7 rather than T7lac promoter was utilized).

d) Purification of pHisBotB Protein from pHisBotB amp T7lac/Bl21(DE3)Cells

Soluble recombinant BotB protein was purified utilizing NiNTA resin from80 ml of cell lysate generated from cells harvested from a pHisBotBfermentation [using the pHisBotB amp T7lac/Bl21(DE3) strain]. Aspredicted from the small scale results above, the majority of theinduced protein was insoluble. As well, the eluted material wascontaminated with multiple E. coli contaminant proteins. A Coomassieblue-stained SDS-PAGE gel containing extracts derived from pHisBotB ampT7lac/Bl21(DE3) cells before and during purification is shown in FIG.34. In FIG. 34, lane 1 contains broad range protein MW markers (BioRad).Lanes 2-5 contain extracts prepared from pHisBotB amp T7lac/Bl21(DE3)cells grown in fermentation culture; lane 2 contains total protein; lane3 contains soluble protein; lane 4 contains protein which did not bindto the NiNTA column (i.e., the flow-through) and lane 5 contains proteineluted from the NiNTA column.

Similar results were obtained using a small scale IDA column utilizing acell lysate from the pHisBotB kan lacIq T7 fermentation described above.250 mls of a 20% w/v PEI clarified lysate (50 gms cell pellet) of botBkan lacIq T7/Bl21(DE3) cells were purified on a small scale IDA column.The total yield of eluted protein was 21 mg protein (assuming 1 mg/mlsolution=2 OD₂₈₀/ml). When analyzed by SDS-PAGE and Coomassie staining,the BotB protein was found to comprise approximately 50% of the elutedprotein with the remainder being a ladder of E. coli proteins similar tothat observed with the NiNTA purification.

The NiNTA alkaline phosphatase conjugate was utilized to detecthis-tagged proteins on a Western blot containing total, soluble, soluble(PEI clarified), soluble (after IDA column) and elution samples from theIDA column purification. The results demonstrated that a smallpercentage of BotB protein was soluble, that the soluble protein was notprecipitated by PEI treatment and was quantitatively bound by the IDAcolumn. Since a 1 liter fermentation harvest yielded a 67.5 gm cellpellet, this indicated that the yield of soluble affinity purified BotBprotein from the IDA column was 14 mg/liter.

Example 39 Co-Expression of Recombinant BotB Proteins and FoldingChaperones in Fermentation Cultures

Fermentations were performed to determine if the simultaneousoverexpression of folding chaperones (i.e., the Gro operon) and the BotBprotein resulted in enhanced solubility of the BotB protein. Thisexample involved fermentation of the pHisBotBkan lacIq T7lac/pACYCGroBL21(DE3), pHisBotB kan T7lac/pACYCGro Bl21(DE3) and pHisBotBkan lacIqT7/pACYCGro BL21(DE3) cell lines. Fermentation was carried out asdescribed in Example 31; 34 μg/ml chloramphenicol was included in thefeeder and fermentation cultures.

a) Fermentation of pHisBotBkan lacIq T7lac/pACYCGro BL21(DE3) Cells

Induction was with 4 gms IPTG at 1 hr 15 min post initiation of theglucose feed. The OD₆₀₀ was 38 at time of induction, then 50, 58.5, 62and 68 at 1-4 hrs post induction. Viable colony counts decreased duringinduction (24, 0, 0, 2, 0 at 0-4 hr induction; dilution 3 utilized 3 μlof dilution 2 cells). Of 24 colonies scored at the time of induction, 24retained the BotB plasmid (kan resistant), 24 contained the chaperoneplasmid (chloramphenicol resistant) and no colonies at induction grew onIPTG+Kan plates (no mutations detected).

Total and soluble extracts were resolved on 12.5% SDS-PAGE gels and wereeither stained with Coomassie blue or subjected to Western blotting(his-tagged proteins were detected utilizing the NiNTA-alkalinephosphatase conjugate). This analysis revealed that the Gro chaperoneswere induced to high levels, but very low level expression of solubleBotB protein was observed, increasing from 1 to 4.0 hrs post induction(no expression detected in uninduced cells, induced protein detectedonly on Western blot). The dramatically lower expression of BotB proteinin the presence of chaperone may be due to promoter occlusion (i.e., thestronger T7 promoter on the chaperone plasmid was preferentiallyutilized).

b) Fermentation of pHisBotB kan T7lac/pACYCGro/Bl21(DE3) Cells

Induction was with 4 gms IPTG at 1 hr post initiation of the glucosefeed. The OD₆₀₀ was 33.5 at time of induction, then 44, 51, 58.5 and 69at 1-4 hrs post induction. Viable colony counts decreased after 2 hrsinduction (43, 65, 74, 0 (70), 0 (70) at 0-4 hr induction; bracketednumbers represent microcolonies; dilution 3 utilized 3 μl of dilution 2cells). Most colonies at induction retained the BotB plasmid (kanresistant) and the chaperone plasmid (chloramphenicol resistant) and nocolonies at induction grew on IPTG+Kan plates (no mutations detected).

Total and soluble extracts were resolved on a 12.5% SDS-PAGE gel andsubjected to Western blotting; his-tagged proteins were detectedutilizing the NiNTA-alkaline phosphatase conjugate. This analysisrevealed that the Gro chaperones were induced to high levels and lowlevel expression of soluble Bot B protein was observed, increasing from1 to 4.0 hrs post induction (no expression detected in uninduced cells).

A small scale IDA purification of BotB protein from a 250 ml PEIclarified 15% w/v extract (37.5 gm cell pellet) yielded approximately12.5 mg protein, of which approximately 50% was BotB protein and 50% wasGroEL chaperone (assessed by Coomassie staining of a 10% SDS-PAGE gel).The NiNTA alkaline phosphatase conjugate was utilized to detecthis-tagged proteins on a Western blot containing total, soluble, soluble(PEI clarified), soluble (after IDA column) and elution samples from theIDA column purification. The results demonstrated that all of the BotBprotein produced by the pHisBotB kan T7lac/pACYCGro/Bl21(DE3) cells wassoluble; the BotB protein was not precipitated by PEI treatment and wasquantitatively bound by the IDA column. Since a 1 liter fermentationharvest yielded a 75 gm cell pellet, this indicated that the yield ofsoluble affinity purified BotB protein from this fermentation was 12.5mg/liter. These results also demonstrated that additional purificationsteps are necessary to separate the chaperone proteins from the BotBprotein.

c) Fermentation of pHisBotBkan lacIq T7/pACYCGro/BL21(DE3) Cells

Induction was with 4 gms IPTG at 2 hr post initiation of the glucosefeed. The OD₆₀₀ was 46 at time of induction, then 56, 63, 69 and 71.5 at1-4 hrs post induction. Viable colony counts decreased after induction(58, 3(5), 3, 0, 0 at 0-4 hr induction; bracketed numbers representmicrocolonies; dilution 3 utilized 3 μL of dilution 2 cells). All(53/53) colonies scored at the time of induction retained the BotBplasmid (kan resistant) and the chaperone plasmid (chloramphenicolresistant) and no colonies at induction grew on IPTG+Kan plates (nomutations detected).

Total and soluble extracts were resolved on a 10% SDS-PAGE gels andWestern blotted and his-tagged proteins were detected utilizing theNiNTA-alkaline phosphatase conjugate. This analysis revealed that theGro chaperones were induced to high levels (observed by ponceau Sstaining), and a much higher expression of soluble BotB protein(compared to expression in the pHisBotB kan T7lac/pACYCGro fermentation)was observed at all timepoints, including uninduced cells (some increasein BotB protein levels were observed after induction).

A small scale IDA purification of BotB protein from a 100 ml PEIclarified 15% w/v extract (15 gm cell pellet) yielded approximately 40mg protein, of which approximately 50% was BotB protein and 50% wasGroEL chaperone, as assessed by Coomassie staining of a 10% SDS-PAGEgel. The NiNTA alkaline phosphatase conjugate was utilized to detecthis-tagged proteins on a Western blot containing total, soluble, soluble(PEI clarified), soluble (after IDA column) and elution samples from theIDA column purification. The results demonstrated that a significantpercentage (i.e., ˜10-20%) of BotB protein was soluble, that thesolubilized protein was not precipitated by PEI treatment and wasquantitatively bound by the IDA column. Since a 10 liter fermentationyielded a 108 gm cell pellet, this indicated that the yield of solubleaffinity purified BotB protein from this fermentation was 144 mg/liter.

In a scale up experiment, 2 liters of a 20% w/v PEI clarified lysate ofpHisBotB kan lacIq T7/pACYCGro/BL21(DE3) cells were purified on a largescale IDA column. The purification was performed in duplicate. The totalyield of BotB protein was 220 and 325 mgs protein in the two experiments(assuming 1 mg/ml solution=2.0 OD₂₈₀/ml). This represents 0.7% or 1.0%,respectively, of the total soluble cellular protein (assuming a PEIlysate having a concentration of 8 mg protein/ml and that the elutedmaterial comprises a 1:1 mixture of BotB and folding chaperone). TheNiNTA alkaline phosphatase conjugate was utilized to detect his-taggedproteins on a Western blot containing total, soluble, soluble (PEIclarified), soluble (after IDA column) and elution samples from the IDAcolumn purification. These results demonstrated that a significantpercentage (i.e., ˜10-20%) of the BotB protein was soluble, that thesolubilized protein was not precipitated by PEI treatment and wasquantitatively bound by the IDA column. Since a 1 liter fermentationharvest yielded a 108 gm cell pellet, this indicated that the yield ofsoluble affinity purified BotB protein from the large scale purificationwas 60 mg or 89 mg/liter. These results also demonstrated that furtherpurification would be necessary to remove the contaminating chaperoneprotein.

The above results provide methodologies for the purification of solubleBotB protein from fermentation cultures, in a form contaminatedpredominantly with a single E. coli protein (the folding chaperoneutilized to enhance solubility). In the next example, methods areprovided for the removal of the contaminating chaperone protein.

Example 40 Removal of Contaminating Folding Chaperone Protein fromPurified Recombinant C. botulinum Type B Protein

In this example size exclusion chromatography and ultrafiltration wasused to purify recombinant BotB protein from the folding chaperones andimidazole in IDA-purified material.

To enhance the solubility of the recombinant BotB protein duringscale-up, the protein was co-expressed with folding chaperones (see Ex.39). During the Ni-IDA purification step, the folding chaperonesco-eluted with the BotB protein in 800 mM imidazole; therefore, a secondpurification step was required to isolate the BotB protein free offolding chaperones. Lane 3 of FIG. 35 contains proteins eluted from anIDA column to which a lysate of pHisBotB kan lacIq T7/pACYCGro/BL21(DE3)cells had been applied; the proteins were resolved on a 4-15%polyacrylamide pre-cast gradient gel (Bio-Rad, Hercules, Calif.) rununder native conditions and then stained with Coomassie blue. In FIG.35, lanes 1 and 4 contain proteins present in peak 1 and peak 2 from aSephacryl S-100 column run as described below; lane 2 is blank.

As seen in lane 3 of FIG. 35, the IDA-purified sample consists primarilyof the folding chaperones and the BotB protein. The fact that thechaperones and the BotB antigen appear as two distinct bands undernative conditions suggested they were not complexed together andtherefore, it should be possible to separate them, using either agradient of imidazole concentrations or size exclusion methods.

In order to determine whether a gradient of imidazole concentrationscould be used to separate the chaperone from the BotB protein, a stepgradient using imidazole at 200, 400, 600, and 800 mM in 50 mM sodiumphosphate, 0.5 M NaCl and 10% glycerol, pH 6.8 was applied to an IDAcolumn (containing proteins bound from a lysate of pHisBotB kan lacIqT7/pACYCGro/BL21(DE3) cells). By narrowing the range of imidazoleconcentrations, it was hoped that the BotB and chaperone proteins woulddifferentially elute at different concentrations of imidazole. Elutedproteins were monitored by absorbance at 280 nm and collected eithermanually or with a fraction collector (BioRad). Protein was found toelute at 200 and 400 mM imidazole only.

FIG. 36 shows a Coomassie stained SDS-PAGE gel containing protein elutedduring the imidazole step gradient. Lane 1 contains broad range MWmarkers (BioRad). Lane 2 contains BotB protein purified by IDAchromatography of an extract of pHisBotB/BL21(DE3) pLysS cells grown inshaker flask culture (i.e., no co-expression of chaperones; Ex. 35).Lane 3 contains a 20% w/v PEI clarified lysate of pHisBotB kan lacIqT7/pACYCGro/BL21(DE3) cells (i.e., the lysate prior to purification byIDA chromatography). Lanes 4 and 5 contain protein which eluted at 200or 400 mM imidazole, respectively. Lane 6 is blank. Lanes 7 and 8contain ⅕ the load present in lanes 4 and 5.

As shown in FIG. 36, both the chaperone and the BotB protein eluted in200 mM imidazole, and more chaperone elutes in 400 mM imidazole, howeverno concentration of imidazole tested permitted the elution of BotBprotein alone. Consequently, no significant purification was achievedusing imidazole at these concentrations.

Because of the considerable difference in molecular weights between thefolding chaperone, which is a multimer with a total molecular weightaround 400 kD (as determined on a Shodex KB 804 sizing column by HPLC),and the recombinant BotB protein (molecular weight around 50 kD), sizeexclusion chromatography was next examined for the ability to separatethese proteins.

a) Size Exclusion Chromatography

A column containing Sephacryl S-100 HR(S-100) (Pharmacia) was poured(2.5 cm×24 cm; ˜110 ml bed volume). The column was equilibrated in abuffer consisting of phosphate buffered saline (10 mM potassiumphosphate, 150 mM NaCl, pH 7.2) and 10% glycerol (Mallinkrodt).Typically, 5 ml of the IDA-purified BotB protein was filtered through a0.45μ syringe filter and applied to the column, and the equilibrationbuffer was pumped through the column at a flow rate of 1 ml/minute.Eluted proteins were monitored by absorbance at 280 nm and collectedeither manually or with a fraction collector. Appropriate tubes werepooled, if necessary, and the protein was quantitated by absorbance at280 nm and/or by BCA protein assay. The isolated peaks were thenanalyzed by native and/or SDS-PAGE to identify the protein and evaluatethe purity.

Because of its larger size, the folding chaperone eluted first, followedby the recombinant BotB protein. A smaller third peak was observed whichfailed to stain when analyzed by SDS-PAGE and therefore was presumed tobe imidazole.

SDS-PAGE analysis (12.5% polyacrylamide, reduced samples) was used toevaluate the purity of the IDA-purified recombinant BotB protein beforeand after S-100 purification. The results are shown in FIG. 33.

In FIG. 33, lane 1 contains broad range MW markers (BioRad). Lane 5contains IDA-purified BotB protein. Lane 6 contains IDA-purified BotBprotein following S-100 purification. Lane 7 is blank (lanes 2-4 werediscussed in Ex. 34 above).

The results shown in FIG. 33 show that the IDA-purified BotB issignificantly contaminated with the folding chaperone (molecular weightabout 60 kD under reducing conditions; lane 6). Following S-100purification, the amount of folding chaperone present in the BotB samplewas reduced dramatically (lane 7). Visual inspection of the Coomassiestained SDS-PAGE gel revealed that after S-100 purification, >90% of thetotal protein present was BotB.

The IDA-purified BotB and the S-100-purified BotB samples were analyzedby HPLC on a size exclusion column (Shodex KB 804); this analysisrevealed that the BotB protein represented 64% of the total protein inthe IDA-purified sample and that following S-100 purification, the BotBprotein represented >95% of the total protein in the sample.

The IDA-purified BotB material was also applied to a ACA 44 (SpectraPor,Houston, Tex.) column. The ACA 44 resin is equivalent to the S-100 resinand chromatography using the ACA 44 resin was carried out exactly asdescribed above for the S-100 resin. The ACA 44 resin was found toseparate the recombinant BotB protein from the folding chaperone. TheACA 44-purified BotB sample was analyzed for endotoxin using the LALassay (Associates of Cape Cod) as describe in Example 24. Two aliquotsof the ACA 44-purified BotB preparation were analyzed and were found tocontain either 58 to 116 EU/mg recombinant protein or 94 to 189 EU/mgrecombinant protein.

These results demonstrate that size exclusion chromatography can be usedto purify the recombinant BotB protein from the folding chaperone andimidazole in IDA-purified material

b) Ultrafiltration for the Separation of Recombinant BotB Protein andChaperones

Ultrafiltration was examined as an alternative method for the separationrecombinant BotB protein and folding chaperones in IDA-purifiedmaterial. While in this example only mixtures of BotB and chaperoneswere separated by ultrafiltration, this technique is suitable for usewith recombinant BotA and BotE proteins as well provided that the washbuffers used are altered as necessary to take into account differentrequirements for solubility.

The recombinant BotB protein and folding chaperones were separated usinga two-step sequential ultrafiltration method. The first membrane usedhad a nominal molecular weight cutoff (MWCO) of approximately 100 kD;this membrane retains the larger folding chaperone while allowing thesmaller recombinant protein to pass through. The addition of severalvolumes of wash buffer may be required to efficiently wash therecombinant protein through the membrane. The second step utilized amembrane with a nominal MWCO of approximately 10 kD. During this step,the recombinant antigen was retained by the membrane and could beconcentrated to the degree desired and the imidazole and excess washbuffer passed through the membrane.

Twenty-seven milliliters of an IDA-purified BotB preparation wasultrafiltered through a 47 mm YM 100 (100 kD MWCO) membrane (Amicon) ina 50 ml stirred cell (Amicon). The membrane was washed in dd H₂O priorto use as recommended by the manufacturer. Six volumes of 10% glycerolin PBS were washed through to remove most of the recombinant BotBprotein and this wash was collected in a separate vessel. The resultingBotB protein-rich filtrate was then concentrated 12-fold using a YM 10(10 kD MWCO) membrane (Amicon), to a final volume of 14 ml. The YM 100and YM 10 concentrates were analyzed along with the lysate startingmaterial by native PAGE using a 4-15% pre-cast gradient gel (BioRad).The results are shown in FIG. 37.

In FIG. 37, lane 1 contains IDA-purified BotB derived from a shakerflask culture (i.e., no co-expression of chaperones; Ex. 35); lane 2contains a 20% w/v PEI clarified lysate of pHisBotB kan lacIqT7/pACYCGro/BL21(DE3) cells; lane 3 shows the lysate of lane 3 after IDApurification; lane 4 contains the YM 10 concentrate and lane 5 containsthe YM 100 concentrate.

The results shown in FIG. 37 demonstrate that the recombinant BotBprotein can be purified away from the folding chaperone byultrafiltration through a 100 kD MWCO membrane (lane 4), leaving thechaperone protein in the 100 kD concentrate (lane 5). Analysis of thesample in lane 5 also showed that very little of the BotB protein wasretained by the 100 kD MWCO membrane after 6 volumes of wash buffer hadbeen applied.

The BotB samples following IDA chromatography and followingultrafiltration through the YM 100 membrane were analyzed by HPLC on asize exclusion column (Shodex KB 804); this analysis revealed that theBotB protein represented 64% of the total protein in the IDA-purifiedsample and that following ultrafiltration through the YM 100 membrane,the BotB protein represented >96% of the total protein in the sample.

The BotB protein purified by ultrafiltration through the YM 100 membranewas examined for endotoxin using the LAL assay (Associates of Cape Cod)as described in Example 24. Two aliquots of the YM 100-purified BotBpreparation were analyzed and were found to contain either 18 to 36EU/mg recombinant protein or 125 to 250 EU/mg recombinant protein.

The above results demonstrate that size exclusion chromatography andultrafiltration can be used to purify recombinant botulinal toxinproteins away from folding chaperones.

Example 41 Cloning and Expression of the C Fragment of the C. botulinumSerotype E Toxin Gene

The C. botulinum type E neurotoxin gene has been cloned and sequencedfrom several different strains [Poulet et al. (1992) Biochem. Biophys.Res. Commun. 183:107 (strain Beluga); Whelan et al. (1992) Eur. J.Biochem. 204:657 (strain NCTC 11219); Fujii et al. (1990) Microbiol.Immunol. 34:1041 (partial sequence of strains Mashike, Iwani and Otaru)and Fujii et al. (1993) J. Gen. Microbiol. 139:79 (strain Mashike)]. Thenucleotide sequence of the type E toxin gene is available from the EMBLsequence data bank under accession numbers X62089 (strain Beluga) andX62683 (strain NCTC 11219). The nucleotide sequence of the coding region(strain Beluga) is listed in SEQ ID NO:49. The amino acid sequence ofthe C. botulinum type E neurotoxin derived from strain Beluga is listedin SEQ ID NO:50. The nucleotide sequence-of the coding region (strainNCTC 11219) is listed in SEQ ID NO:51. The amino acid sequence of the C.botulinum type E neurotoxin derived from strain NCTC 11219 is listed inSEQ ID NO:52.

The DNA sequence encoding the native C. botulinum serotype E C fragmentgene derived from the Beluga strain can be expressed as ahistidine-tagged protein using the pETHisb vector; the resulting codingregion is listed in SEQ ID NO:53 and the corresponding amino acidsequence is listed in SEQ ID NO:54. The DNA sequence encoding the Cfragment of the native C. botulinum serotype E gene derived from theNCTC 11219 strain can be expressed as a histidine-tagged fusion proteinusing the pETHisb vector; the resulting coding region is listed in SEQID NO:55 and the corresponding amino acid sequence is listed in SEQ IDNO:56. The C fragment region from any strain of C. botulinum serotype Ecan be amplified and expressed using the approach illustrated belowusing the C fragment derived from C. botulinum type E 2231 strain (ATCC#17786).

The type E neurotoxin gene is synthesized as a single polypeptide chainwhich may be converted to a double-chain form (i.e., a heavy chain and alight chain) by cleavage with trypsin; unlike the type A neurotoxin, thetype E neurotoxin exists essentially only in the single-chain form. The50 kD carboxy-terminal portion of the heavy chain is referred to as theC fragment or the H_(C) domain. Expression of the C fragment of C.botulinum type E toxin in heterologous hosts (e.g., E. coli) has notbeen previously reported.

The native C fragment of the C. botulinum serotype E toxin (BotE) genewas cloned and inserted into expression vectors to facilitate expressionof the recombinant BotE protein in E. coli. This example involved PCRamplification of the gene, cloning, and construction of expressionvectors.

The BotE serotype gene was isolated using PCR as described for the BotAserotype gene in Example 28. The C. botulinum type E strain was obtainedfrom the American Type Culture Collection (ATCC #17786; strain 2231).The following primer pair was used in the PCR amplification:5′-CGCCATGGCTCTTTCTTCTTAT ACAGATGAT-3′ (5′ primer, engineered NcoI siteunderlined) (SEQ ID NO:57) and 5′-GCAAGCTTTTATTTTTCTTGCCATCCATG-3′ (3′primer, engineered HindIII site underlined, native gene terminationcodon italicized) (SEQ ID NO:58). The PCR product was inserted intopCRscript as described in Example 28. The resulting pCRscript BotE clonewas confirmed by restriction digestion, as well as, by obtaining thesequence of approximately 300 bases located at the 5′ end of the Cfragment coding region using standard DNA sequencing methods. Theresulting BotE sequence was identical to that of the published C.botulinum type E toxin sequence [Whelan et al (1992), supra].

The NheI(filled)/HindIII fragment from a pCRscript BotE recombinant wascloned into pETHisb vector as described for BotA C fragment in Example28. The resulting construct was termed pHisBotE. pHisBotE expresses theBotE gene under the control of the T7 lac promoter and the resultingprotein contains an N-terminal 10×His-tag affinity tag.

The pHisBotE expression construct was transformed into BL21(DE3) pLysScompetent cells and 1 liter cultures were grown, induced and his-taggedproteins were purified utilizing a NiNTA resin (eluted in low pH elutionbuffer) as described in Example 28. Total, soluble and purified proteinswere resolved by SDS-PAGE and detected by Coomassie staining. Theresults are shown in FIG. 38.

In FIG. 38, lane 1 contains broad range MW markers (BioRad); lane 2contains a total protein extract; lane 3 contains a soluble proteinextract; lane 4 contains proteins present in the flow through from theNiNTA column (this sample was not diluted prior to loading and thereforerepresents a load 5× that of the load applied for the total and solubleextracts in lanes 2 and 3); lane 5 contains proteins eluted from theNiNTA column; lane 6 contains protein eluted from a NiNTA column whichhad been stored at −20° C. for 1 year.

The pHisBotE protein was expressed at moderate levels (7 mg/liter) as atotally soluble protein. The purified protein migrated as a single bandof the predicted MW.

Western blot hybridization utilizing a chicken anti-C. botulinumserotype E toxoid primary antibody (generated by immunization of hens asdescribed in Example 3 using C. botulinum serotype E toxoid) was alsoperformed on the total, soluble and purified BotE proteins. Samples ofBotA and BotB C fragments were also included on the gels to facilitateMW and immunogenicity comparisons. Strong immunoreactivity was detectedusing the anti-C. botulinum type E toxoid antibody only with the BotEprotein.

These results demonstrate that the native BotE gene sequences can beexpressed as a soluble his-tagged protein in E. coli and purified bymetal-chelation affinity chromatography.

Example 42 Generation of Neutralizing Antibodies Using the RecombinantpHisBotE Protein

The ability of the purified pHisBotE protein to generate neutralizingantibodies was examined. Nine BALBc mice were immunized with BotEprotein (purified as described in Ex. 41) using Gerbu GMDP adjuvant (CCBiotech). The low pH elution was mixed with Gerbu adjuvant and used toimmunize mice. Each mouse received a subcutaneous injection of 100 μlantigen/adjuvant mix (35 μg antigen+1 μg adjuvant) on day 0. Mice weresubcutaneously boosted as above on day 14 and bled on day 28. Mice weresubsequently boosted and bled on day 70.

Anti-C. botulinum serotype E toxoid titers were determined in day 28serum from individual mice from each group using the ELISA protocoloutlined in Example 29 with the exception that the plates were coatedwith C. botulinum serotype E toxoid, and the primary antibody was achicken anti-C. botulinum serotype E toxoid. Seroconversion [relative tocontrol mice immunized with the p6×HisBotA antigen (Ex. 29)] wasobserved with all 9 mice immunized with the purified pHisBotE protein.

The ability of the anti-BotE antibodies to neutralize native C.botulinum type E toxin was tested in a mouse-C. botulinum neutralizationmodel using pooled mouse serum (see Ex. 23b). The LD₅₀ of purified C.botulinum type E toxin complex (Dr. Eric Johnson, University ofWisconsin, Madison) was determined by a intraperitoneal (IP) method[Schantz and Kautler (1978), supra] using 18-22 g female ICR mice. Theamount of neutralizing antibodies present in the serum of the immunizedmice was determined using serum antibody titrations. The various serumdilutions (0.01 ml) were mixed with 5 LD₅₀ units of C. botulinum type Etoxin and the mixtures were injected IP into mice. The neutralizationswere performed in duplicate. The mice were then observed for signs ofbotulism for 4 days. Undiluted serum from day 28 did not protect, whileundiluted, 1/10 diluted and 1/100 diluted day 70 serum protected (1005of animals) while 1/1000 diluted day 70 serum did not. This correspondsto a neutralization titer of 50-500 IU/ml.

These results demonstrate that seroconversion occurred and neutralizingantibodies were induced when the recombinant BotE protein was utilizedas the immunogen.

Example 43 Construction of Vectors to Facilitate Expression ofHis-Tagged BotE Protein in Fermentation Cultures

A number of expression vectors were constructed to facilitate theexpression of recombinant BotE protein in large scale fermentationculture. These constructs varied as to the strength of the promoterutilized (T7 or T7lac) and the presence of repressor elements (lacIq) onthe plasmid. The resulting constructs varied in the level of expressionachieved and in plasmid stability which facilitated the selection of anoptimal expression system for fermentation scaleup. This exampleinvolved a) construction of BotE expression vectors and b) determinationof expression levels in small scale cultures.

a) Construction of BotE Expression Vectors

The BotE expression vectors created for fermentation culture wereengineered to utilize the kanamycin rather than the ampicillinresistance gene, and contained either the T7 or T7lac promoter, with orwithout the lacIq gene for the reasons outlined in Example 30.

In all cases, the protein expressed by the various expression vectors isthe pHisBotE protein described in Example 41, with the only differencesbetween clones being the alteration of various regulatory elements.Using the designations outlined below, the pHisBotE clone (Ex. 41) isequivalent to pHisBotE amp T7lac.

i) Construction of pHisBotE kan lacIq T7lac

pHisBotE kan lacIq T7lac was constructed by inserting the XbaI/HindIIIfragment of pHisBotE which contains the BotE gene sequences intoXbaI/HindIII-cleaved pET24a vector. Proper construction was confirmed byrestriction digestion.

ii) Construction of pHisBotE kan T7

pHisBotE kan T7 was constructed by ligating the BotE-containingXbaI/SapI fragment of pHisBotE kan lacIqT7lac to the T7promoter-containing XbaI/SapI fragment of pET23a. Proper constructionwas confirmed by restriction digestion.

iii) Construction of pHisBotE kan lacIqT7

pHisBotE kan lacIqT7 was constructed by inserting the BglII/HindIIIfragment from pHisBotE kan T7 which contains the BotE gene sequencesinto BglII/HindIII-cleaved pET24 vector. Proper construction wasconfirmed by restriction digestion.

b) Determination of BotE Expression Levels in Small Scale Cultures

The three BotE kan expression vectors described above were transformedinto Bl21(DE3) competent cells and 50 ml (2XYT+40 μg/ml kan) cultureswere grown and induced with ITPG as described in Example 28. Total andsoluble protein extracts from before and after induction made asdescribed in Example 28. The total and soluble extracts were resolved ona 12.5% SDS-PAGE gel, and his-tagged proteins were detected on a Westernblot utilizing the NiNTA-alkaline phosphatase conjugate as described inExample 31(c)(iii). The results showed that all three BotE cell linesexpressed his-tagged proteins of the predicted MW for the BotE proteinupon induction. The results also demonstrated that the two constructsthat contained the T7 promoter expressed the BotE protein beforeinduction, while the T7lac promoter construct did not. Upon induction,the T7 promoter-containing constructs induced to higher levels than theT7lac-containing construct, with the pHisBotE kan lacIqT7/Bl21(DE3)cells accumulating the maximal levels of BotE protein.

Example 44 Expression and Purification of pHisBotE from FermentationCultures

Based on the small scale inductions performed in Example 43, thepHisBotE kan lacIq T7/Bl21(DE3) strain was selected for fermentationscaleup. This example involved the fermentation and purification ofrecombinant BotE C fragment protein.

A fermentation with the pHisBotE kan lacIq T7/Bl21(DE3) strain wasperformed as described in Example 31. The fermentation culture wasinduced 2 hrs post start of the glucose feed with 4 gm IPTG (finalconcentration=1.6 mM). The OD₆₀₀ was 42 at time of induction, then 46.5,48, 53 and 54 at 1-4 hrs post induction. Viable colony counts decreasedfrom 0-4 hr induction [131, 4 (28), 7 (3), 7, 8; dilution 3 utilized 6μl of dilution 2 cells; bracketed colonies are microcolonies]. All(32/32) colonies scored at the time of induction retained the BotEplasmid (kan resistant) and no colonies at induction grew on IPTG+Kanplates (no mutations detected). These results were indicative of strongpromoter induction, since colony viability reduced after induction, andthe culture stopped growing during fermentation (stopped at 54 OD₆₀₀ml).

Total and soluble extracts were resolved on a 12.5% SDS-PAGE gel andtotal protein was detected by staining with Coomassie blue. The resultsare shown in FIG. 39.

In FIG. 39, lane 1 contains total protein from a pHisBotA kan T7lac/Bl21(DE3) pLysS fermentation (Ex. 24). Lanes 2-9 contain extractsprepared from the above pHisBotE kan lacIq T7/Bl21(DE3) fermentation;lanes 2-4 contain total protein extracts prepared at 0, 1 and 2 hourspost-induction, respectively. Lane 5 contains a soluble protein extractprepared at 2 hours post-induction. Lanes 6 and 7 contain total andsoluble extracts prepared at 3 hours post-induction, respectively. Lanes8 and 9 contain total and soluble extracts prepared at 4 hourspost-induction, respectively. Lane 10 contains broad range MW markers(BioRad).

The results shown in FIG. 39 demonstrate that moderate level inductionof totally soluble Bot E protein was observed, increasing from 1 to 4hrs post induction (no expression was detected in uninduced cells). Froma 2 liter fermentation harvest a 155 gm (wet wt) cell pellet wasobtained and used to make a PEI-clarified lysate (1 liter in CRB, pH6.8). The lysate was applied to a large scale IDA column and 200 mg ofBotE protein, which was found to be greater than 95% pure (as judged byvisual inspection of a Coomassie stained SDS-PAGE gel), was recovered.This represents 2.5% of the total soluble cellular protein (assuming aPEI lysate having a concentration of 8 mg protein/ml) and corresponds toa yield of 100 mg BotE protein/liter of fermentation culture.

The above results demonstrate that high levels of the recombinant BotEprotein can be expressed and purified from fermentation cultures.

Example 45 Removal of Imidazole from Purified Recombinant BotE ProteinPreparations

The expression of recombinant BotE protein, unlike the BotA and BotBproteins, did not require the presence of folding chaperones to maintainsolubility during scale-up. A size exclusion chromatography step wasincluded however to remove the imidazole from the sample and exchangethe IDA elution buffer for one consistent with the BotA antigen.

A Sephacryl S-100 HR(S-100; Pharmacia) column was poured (2.5 cm×24 cm;bed volume ˜110 ml). Under these conditions, the BotE protein should beretained by the beads to a lesser degree than the smaller imidazole,therefore the BotE protein should elute from the column before theimidazole. The column was equilibrated in a buffer consisting of 50 mMsodium phosphate, 0.5 M NaCl, and 10% glycerol (all reagents fromMallinkrodt). Five milliliters of the IDA-purified BotE protein (Ex. 44)was filtered through a 0.45μ syringe filter and applied to the S-100column, and equilibration buffer was pumped through the column at a flowrate of 1 ml/minute. Eluted proteins were monitored by absorbance at 280nm, and collected either manually or with a fraction collector.Appropriate tubes were pooled if necessary, and the protein wasquantitated by absorbance at 280 nm and/or BCA protein assay. Theisolated peaks were then analyzed by native and/or SDS-PAGE to identifythe protein(s) and evaluate the purity.

FIG. 40 provides a representative chromatogram showing the purificationof IDA-purified BotE on the S-100 column. Even though folding chaperoneswere not over-expressed with this antigen, a small amount of proteineluted at a time consistent with the folding chaperones expressed withBotA and BotB proteins (Gro) (see the first peak). The second peak inthe chromatogram contained the BotE protein, and the third peak waspresumably imidazole. This presumed imidazole peak was isolated incomparable levels in IDA-purified BotA and BotB protein preparations aswell.

These results demonstrate that size exclusion chromatography can be usedto remove imidazole and traces of contaminating high molecular weightproteins from IDA-purified BotE protein preparations.

The S-100-purified BotE protein was tested for endotoxin contaminationusing the LAL assay as described in Example 24. This preparation wasfound to contain 64 to 128 EU/mg recombinant protein and is thereforesubstantially free of endotoxin.

The S-100 purified BotE was mixed with purified preparations of BotA andBotB proteins and used to immunize mice; 5 μg of each Bot protein wasused per immunization and alum was included as an adjuvant. After twoimmunizations with this trivalent vaccine, the immunized mice werechallenged with C. botulinum toxin. The immunized mice containedneutralizing antibodies sufficient to neutralize between 100,000 to1,000,000 LD₅₀ of either toxin A or toxin B and between 1,000 to 10,000LD₅₀ of toxin E. The titer of neutralizing antibodies directed againsttoxin E would be expected to increase following subsequent boosts withthe vaccine. These results demonstrate that a trivalent vaccinecontaining recombinant BotA, BotB and BotE proteins provokesneutralizing antibodies.

Example 46 Expression of the C Fragment of the C. botulinum Serotype CToxin Gene and Generation of Neutralizing Antibodies

The C. botulinum type C1 neurotoxin gene has been cloned and sequenced[Kimura et al. (1990) Biochem. Biophys. Res. Comm. 171:1304]. Thenucleotide sequence of the toxin gene derived from the C. botulinum typeC strain C-Stockholm is available from the EMBL/GenBank sequence databanks under the accession number D90210; the nucleotide sequence of thecoding region is listed in SEQ ID NO:59. The amino acid sequence of theC. botulinum type C1 neurotoxin derived from this strain is listed inSEQ ID NO: 60.

The DNA sequence encoding the native C. botulinum serotype C₁-C fragmentgene derived from the C-Stockholm strain can be expressed using thepETHisb vector; the resulting coding region is listed in SEQ ID NO:61and the corresponding amino acid sequence is listed in SEQ ID NO:62. TheC fragment region from any strain of C. botulinum serotype C can beamplified and expressed using the approach illustrated below using the Cfragment derived from C. botulinum type C C-Stockholm strain. Expressionof the C fragment of C. botulinum type C1 toxin in heterologous hosts(e.g., E. coli) has not been previously reported.

The C fragment of the C. botulinum serotype C1 (BotC1) toxin gene iscloned using the protocols and conditions described in Example 28 forthe isolation of the native BotA gene. A number of C. botulinum serotypeC strains (expressing either or both C1 and C2 toxin) are available fromthe ATCC [eg., 2220 (ATCC 17782), 2239 (ATCC 17783), 2223 (ATCC 17784; atype C-β strain; C-β strains produce C2 toxin), 662 (ATCC 17849; a typeC-α strain; C-α strains produce mainly C1 toxin and a small amount of C2toxin), 2021 (ATCC 17850; a type C-α strain) and VPI 3803 (ATCC 25766)].Alternatively, other type C strains may be employed for the isolation ofsequences encoding the C fragment of C. botulinum serotype C toxin.

The following primer pair is used to amplify the BotC gene:5′-CGCCATGGCTTTATTAAAAGATATAATTAATG-3′ [5′ primer, engineered NcoI siteunderlined (SEQ ID NO:63)] and 5′-GCAAGCTTTTATTCACTTACAGGTAC AAAACC-3′[3′ primer, engineered HindIII site underlined, native gene terminationcodon italicized (SEQ ID NO:64)]. Following PCR amplification, the PCRproduct is inserted into the pCRscript vector and then the 1.5 kbfragment is cloned into pETHisb vector as described for BotA C fragmentgene in Example 28. The resulting construct is termed pHisBotC. Properconstruction is confirmed by DNA sequencing of the BotC sequencescontained within pHisBotC.

pHisBotC expresses the BotC gene sequences under the transcriptionalcontrol of the T7 lac promoter and the resulting protein contains anN-terminal 10×His-tag affinity tag. The pHisBotC expression construct istransformed into BL21(DE3) pLysS competent cells and 1 liter culturesare grown, induced and his-tagged proteins are purified utilizing aNiNTA resin (eluted in 250 mM imidazole, 20% glycerol) as described inExample 28. Total, soluble and purified proteins are resolved bySDS-PAGE and detected by Coomassie staining and Western blothybridization utilizing a Ni-NTA-alkaline phosphatase conjugate (Qiagen)which recognizes his-tagged proteins as described in Example 31(c)(iii). This analysis permits the determination of expression levelsof the pHisBotC protein (i.e., number of mg/liter expressed as a solubleprotein). The purified BotC protein will migrate as a single band of thepredicted MW (i.e., ˜50 kD).

The level of expression of the pHisBotC protein may be modified(increased) by substitution of the T7 promoter for the T7lac promoter,or by inclusion of the lacIq gene on the expression plasmid, and plasmidexpressed in BL21(DE3) cell lines in fermentation cultures as describedin Example 30. If only very low levels (i.e., less than 0.5%) of solublepHisBotC protein are expressed using the above expression systems, thepHisBotC construct may be co-expressed with pACYCGro construct asdescribed in Example 32. In this case, the recombinant BotC protein mayco-purify with the folding chaperones. The contaminating chaperones maybe removed as described in Example 34. Preparations of purified pHisBotCprotein are tested for endotoxin contamination using the LAL assay asdescribed in Example 24.

The purified pHisBotC protein is used to generate neutralizingantibodies. BALBc mice are immunized with the BotC protein using GerbuGMDP adjuvant (CC Biotech) as described in Example 36. The ability ofthe anti-BotC antibodies to neutralize native C. botulinum type C toxinis demonstrated using the mouse-C. botulinum neutralization modeldescribed in Example 36.

Example 47 Expression of the C Fragment of the C. botulinum Serotype DToxin Gene and Generation of Neutralizing Antibodies

The C. botulinum type D neurotoxin gene has been cloned and sequenced[Sunagawa et al. (1992) J. Vet. Med. Sci. 54:905 and Binz et al. (1990)Nucleic Acids Res. 18:5556]. The nucleotide sequence of the toxin genederived from the CB16 strain is available from the EMBL/GenBank sequencedata banks under the accession number S49407; the nucleotide sequence ofthe coding region is listed in SEQ ID NO:65. The amino acid sequence ofthe C. botulinum type D neurotoxin derived from the CB16 strain islisted in SEQ ID NO:66.

The DNA sequence encoding the native C. botulinum serotype D C fragmentgene derived from a BotD expressing strain can be expressed using thepETHisb vector; the resulting coding region is listed in SEQ ID NO:67and the corresponding amino acid sequence is listed in SEQ ID NO:68. TheC fragment region from any strain of C. botulinum serotype D can beamplified and expressed using the approach illustrated below using the Cfragment derived from C. botulinum type D CB16 strain. Expression of theC fragment of C. botulinum type D toxin in heterologous hosts (e.g., E.coli) has not been previously reported.

The C fragment of the C. botulinum serotype D (BotD) toxin gene iscloned using the protocols and conditions described in Example 28 forthe isolation of the native BotA gene. A number of C. botulinum type Dstrains are available from the ATCC [e.g., ATCC 9633, 2023 (ATCC 17851),and VPI 5995 (ATCC 27517)].

The following primer pair is used to amplify the BotD gene:5′-CGCCATGGCTTTATTAAAAGATATAATTAATG-3′ [5′ primer, engineered NcoI siteunderlined (SEQ ID NO:63)] and 5′-GCAAGCTTTTACTCTACCCATCCTGGATCCCT-3′[3′ primer, engineered HindIII site underlined, native gene terminationcodon italicized (SEQ ID NO:69)]. Following PCR amplification, the PCRproduct is inserted into the pCRscript vector and then the 1.5 kbfragment is cloned into pETHisb vector as described for BotA C fragmentgene in Example 28. The resulting construct is termed pHisBotD. pHisBotDexpresses the BotD gene sequences under the transcriptional control ofthe T7 lac promoter and the resulting protein contains an N-terminal10×His-tag affinity tag. The pHisBotD expression construct istransformed into BL21(DE3) pLysS competent cells and 1 liter culturesare grown, induced and his-tagged proteins are purified utilizing aNiNTA resin as described in Example 28. Total, soluble and purifiedproteins are resolved by SDS-PAGE and detected by Coomassie staining andWestern blot hybridization utilizing a Ni-NTA-alkaline phosphataseconjugate (Qiagen) which recognizes his-tagged proteins as described inExample 31 (c)(iii). This analysis permits the determination ofexpression levels of the pHisBotD protein (i.e., number of mg/literexpressed as a soluble protein). The purified BotD protein will migrateas a single band of the predicted MW (i.e., ˜50 kD).

The level of expression of the pHisBotD protein may be modified(increased) by substitution of the T7 promoter for the T7lac promoter,or by inclusion of the lacIq gene on the expression plasmid, and plasmidexpressed in BL21(DE3) cell lines in fermentation cultures as describedin Example 30. If only very low levels (i.e., less than about 0.5%) ofsoluble pHisBotD protein are expressed using the above expressionsystems, the pHisBotD construct may be co-expressed with pACYCGroconstruct as described in Example 32. In this case, the recombinant BotDprotein may co-purify with the folding chaperones. The contaminatingchaperones may be removed as described in Example 34. Preparations ofpurified pHisBotD protein are tested for endotoxin contamination usingthe LAL assay as described in Example 24.

The purified pHisBotD protein is used to generate neutralizingantibodies. BALBc mice are immunized with the BotD protein using GerbuGMDP adjuvant (CC Biotech) as described in Example 36. The ability ofthe anti-BotD antibodies to neutralize native C. botulinum type D toxinis demonstrated using the mouse-C. botulinum neutralization modeldescribed in Example 36.

Example 48 Expression of the C Fragment of the C. botulinum Serotype FToxin Gene and Generation of Neutralizing Antibodies

The C. botulinum type F neurotoxin gene has been cloned and sequenced[East et al. (1992) FEMS Microbiol. Lett. 96:225]. The nucleotidesequence of the toxin gene derived from the 202F strain (ATCC 23387) isavailable from the EMBL/GenBank sequence data banks under the accessionnumber M92906; the nucleotide sequence of the coding region is listed inSEQ ID NO:70. The amino acid sequence of the C. botulinum type Fneurotoxin derived from the 202F strain is listed in SEQ ID NO:71.

The DNA sequence encoding the native C. botulinum serotype F C fragmentgene derived from the 202F strain can be expressed using the pETHisbvector; the resulting coding region is listed in SEQ ID NO:72 and thecorresponding amino acid sequence is listed in SEQ ID NO:73. The Cfragment region from any strain of C. botulinum serotype F can beamplified and expressed using the approach illustrated below using the Cfragment derived from C. botulinum type F 202F strain. Expression of theC fragment of C. botulinum type F toxin in heterologous hosts (e.g., E.coli) has not been previously reported.

The C fragment of the C. botulinum serotype F (BotF) toxin gene iscloned using the protocols and conditions described in Example 28 forthe isolation of the native BotA gene. The C. botulinum type F 202Fstrain is obtained from the American Type Culture Collection (ATCC23387). Alternatively, sequences encoding the BotF toxin may be isolatedfrom any BotF expressing strain [e.g., VPI 4404 (ATCC 25764), VPI 2382(ATCC 27321) and Langeland (ATCC 35415)].

The following primer pair is used to amplify the BotF gene:5′-CGCCATGGCTATTCTAATTATATATTTTAATAG-3′ [5′ primer, engineered NcoI siteunderlined (SEQ ID NO:74)] and 5′-GCAAGCTTTCATTCTTTCCATCCATTCTC-3′ [3′primer, engineered HindIII site underlined, native gene terminationcodon italicized (SEQ ID NO:75)]. Following PCR amplification, the PCRproduct is inserted into the pCRscript vector and then the 1.5 kbfragment is cloned into pETHisb vector as described for BotA C fragmentgene in Example 28. The resulting construct is termed pHisBotF.

pHisBotF expresses the BotF gene sequences under the transcriptionalcontrol of the T7 lac promoter and the resulting protein contains anN-terminal 10×His-tag affinity tag. The pHisBotF expression construct istransformed into BL21(DE3) pLysS competent cells and 1 liter culturesare grown, induced and his-tagged proteins are purified utilizing aNiNTA resin as described in Example 28. Total, soluble and purifiedproteins are resolved by SDS-PAGE and detected by Coomassie staining andWestern blot hybridization utilizing a Ni-NTA-alkaline phosphataseconjugate (Qiagen) which recognizes his-tagged proteins as described inExample 31 (c)(iii). This analysis permits the determination ofexpression levels of the pHisBotF protein (i.e., number of mg/literexpressed as a soluble protein). The purified BotF protein will migrateas a single band of the predicted MW (i.e., ˜50 kD).

The level of expression of the pHisBotF protein may be modified(increased) by substitution of the T7 promoter for the T7lac promoter,or by inclusion of the lacIq gene on the expression plasmid, and plasmidexpressed in BL21(DE3) cell lines in fermentation cultures as describedin Example 30. If only very low levels (i.e., less than about 0.5%) ofsoluble pHisBotF protein are expressed using the above expressionsystems. the pHisBotF construct may be co-expressed with pACYCGroconstruct as described in Example 32. In this case, the recombinant BotFprotein may co-purify with the folding chaperones. The contaminatingchaperones may be removed as described in Example 34. Preparations ofpurified pHisBotF protein are tested for endotoxin contamination usingthe LAL assay as described in Example 24.

The purified pHisBotF protein is used to generate neutralizingantibodies. BALBc mice are immunized with the BotF protein using GerbuGMDP adjuvant (CC Biotech) as described in Example 36. The ability ofthe anti-BotF antibodies to neutralize native C. botulinum type F toxinis demonstrated using the mouse-C. botulinum neutralization modeldescribed in Example 36.

Example 49 Expression of the C Fragment of the C. botulinum Serotype GToxin Gene and Generation of Neutralizing Antibodies

The C. botulinum type G neurotoxin gene has been cloned and sequenced[Campbell et al. (1993) Biochimica et Biophysica Acta 1216:487 and Binzet al. (1990) Nucleic Acids Res. 18:5556]. The nucleotide sequence ofthe toxin gene derived from the 113/30 strain (NCFB 3012) is availablefrom the EMBL/GenBank sequence data banks under the accession numberX74162; the nucleotide sequence of the coding region is listed in SEQ IDNO:76. The amino acid sequence of the C. botulinum type G neurotoxinderived from this strain is listed in SEQ ID NO:77.

The DNA sequence encoding the native C. botulinum serotype G C fragmentgene derived from the 113/30 strain can be expressed using the pETHisbvector; the resulting coding region is listed in SEQ ID NO:78 and thecorresponding amino acid sequence is listed in SEQ ID NO:79. The Cfragment region from any strain of C. botulinum serotype G can beamplified and expressed using the approach illustrated below using the Cfragment derived from C. botulinum type G 113/30 strain. Expression ofthe C fragment of C. botulinum type G toxin in heterologous hosts (e.g.,E. coli) has not been previously reported.

The C fragment of the C. botulinum serotype G (BotG) toxin gene iscloned using the protocols and conditions described in Example 28 forthe isolation of the native BotA gene. The C. botulinum type G 113/30strain is obtained from the NCFB. The following primer pair is used toamplify the BotG gene: 5′-CGCCATGGCTGAC ACAATTTTAATACA AGT-3′ [5′primer, engineered NcoI site underlined (SEQ ID NO:80)] and5′-GCCTCGAGTTATTCTGTCCATCCTTCATCCAC-3′ [3′ primer, engineered XhoI siteunderlined, native gene termination codon italicized (SEQ ID NO:81)].Following PCR amplification, the PCR product is inserted into thepCRscript vector and then the 1.5 kb fragment is cloned into pETHisbvector as described for BotA C fragment gene in Example 28 with theexception that the sequences encoding BotG are excised from thepCRscript vector by digestion with NcoI and XhoI and the NcoI site isblunted (the BotG sequences contain an internal HindIII site). This NcoI(filled)/XhoI fragment is then ligated to the pETHisb vector which hasbeen digested with NheI and Salt and the NheI site is blunted. Theresulting construct is termed pHisBotG.

pHisBotG expresses the BotG gene sequences under the transcriptionalcontrol of the T7 lac promoter and the resulting protein contains anN-terminal 10×His-tag affinity tag. The pHisBotG expression construct istransformed into BL21(DE3) pLysS competent cells and 1 liter culturesare grown, induced and his-tagged proteins are purified utilizing aNiNTA resin as described in Example 28. Total, soluble and purifiedproteins are resolved by SD S-PAGE and detected by Coomassie stainingand Western blot hybridization utilizing a Ni-NTA-alkaline phosphataseconjugate (Qiagen) which recognizes his-tagged proteins as described inExample 31 (c)(iii). This analysis permits the determination ofexpression levels of the pHisBotG protein (i.e., number of mg/literexpressed as a soluble protein). The purified BotG protein will migrateas a single band of the predicted MW (i.e., ˜50 kD).

The level of expression of the pHisBotG protein may be modified(increased) by substitution of the T7 promoter for the T7ac promoter, orby inclusion of the lacIq gene on the expression plasmid, and plasmidexpressed in BL21(DE3) cell lines in fermentation cultures as describedin Example 30. If only very low levels (i.e., less than about 0.5%) ofsoluble pHisBotG protein are expressed using the above expressionsystems, the pHisBotG construct may be co-expressed with pACYCGroconstruct as described in Example 32. In this case, the recombinant BotGprotein may co-purify with the folding chaperones. The contaminatingchaperones may be removed as described in Example 34. Preparations ofpurified pHisBotG protein are tested for endotoxin contamination usingthe LAL assay as described in Example 24.

The purified pHisBotG protein is used to generate neutralizingantibodies. BALBc mice are immunized with the BotG protein using GerbuGMDP adjuvant (CC Biotech) as described in Example 36. The ability ofthe anti-BotG antibodies to neutralize native C. botulinum type G toxinis demonstrated using the mouse-C. botulinum neutralization modeldescribed in Example 36.

Example 50 Expression of Recombinant Botulinal Toxin Proteins inEucaryotic Host Cells

Recombinant botulinal C fragment proteins may be expressed in eucaryotichost cells, such as yeast and insect cells.

a) Expression in Yeast

Botulinal C fragments derived from serotypes A, B, C, D, E, F and G maybe expressed in yeast cells using a variety of commercially availablevectors. For example, the pPIC3K and pPIC9K expression vectors(Invitrogen) may be employed for expression in the methylotrophic yeast,Pichia pastoris. When the pPIC3K vector is employed, expression of thebotulinal C fragment protein will be intracellular. When the pPIC3Kvector is employed, the botulinal C fragment protein will be secreted(the alpha factor secretion signal is provided on the pPIC9K vector).

DNA sequences encoding the desired C fragment is inserted into thesevectors using techniques known to the art. Briefly, the desiredbotulinal expression cassette (including sequences encoding the his-tag;described in the preceding examples) is amplified using the PCR inconjunction with primers that incorporate unique restriction sites atthe termini of the amplified fragment. Suitable restriction enzyme sitesinclude SnaBI, EcoRI, AvrII and NotI. When the botulinal C fragment isto be expressed using the pPIC3K vector, the initiator methionine (ATG)is provided by the desired Bot gene sequence and a Kozak consensussequence is engineered upstream of the ATG (e.g., ACCATGG).

The amplified restriction fragment containing the botulinal C fragmentgene is then cloned into the desired expression vector. Recombinantclones are integrated into the Pichia pastoris genome and recombinantprotein expression is induced using methanol following themanufacturer's instructions (Invitrogen Pichia expression kit manual).

C. botulinum genes are A/T rich and contain multiple sequences that aresimilar to yeast transcriptional termination signals (e.g., TTTTTATA).If premature transcription termination is observed when the botulinal Cfragment genes are expressed in yeast, the transcription terminationsignals present in the C fragment genes can be removed by either sitedirected mutagenesis (utilizing the PALTER system; Promega) or byconstruction of synthetic genes utilizing overlapping synthetic primers.

The botulinal C fragment genes may be expressed in other yeast cellsusing other commercially available vectors [e.g., using the pYES2 vector(Invitrogen) and S. cerevisiae cells (Invitrogen)].

b) Expression in Insect Cells

Botulinal C fragments derived from serotypes A, B, C, D, E, F and G maybe expressed in insect cells using a variety of commercially availablevectors. For example, the pBlueBac4 transfer vector (invitrogen) may beemployed for expression in Spodoptera frugiperda (Sf9) insect cells(baculovirus expression system) (equivalent baculovirus vectors and hostcells are available from other vendors, e.g., Pharmingen, San Diego,Calif.). Botulinal C fragments contained on NcoI/HindIII fragmentscontained within the pHisBotA-G expression constructs (described in thepreceding examples) are cloned into the pBlueBac4 vector (digested withNcoI and HindIII); the NcoI site present on the C fragment constructsoverlaps with the start codon of the fusion proteins. In the case ofbotulinal C fragment clones that contain internal HindIII sites (e.g.,using the BotG sequences described in Ex. 49), the C fragment gene iscontained within a NcoI/XhoI fragment on the pHisBot construct. ThisNcoI/XhoI fragment is excised from pHisBot and inserted into pBlueBac4digested with NcoI and SalI. Recombinant baculoviruses are made and thedesired recombinant C fragment is expressed in Sf9 cells using theprotocols provided by the manufacturer (Invitrogen MaxBac manual). Theresulting constructs will express the pHisBot protein intracellularly(including the N-terminal his-tag) under the control of the polyhedrinpromoter. For extracellular secretion of botulinal C fragment proteins,the C fragment sequences from the pHisBot constructs are cloned into thepMelBacB vector (Invitrogen) as described above for the pBlueBac4vector. When the pMelBacB vector is employed, the his-tagged botulinal Cfragment proteins are secreted (utilizing a vector-encoded honeybeemelittin secretion signal) and contain a nine amino acid extension atthe N-terminus.

His-tagged botulinal C fragments expressed in yeast or insect cells arepurified using metal chelation columns as described in the precedingexamples.

From the above it is clear that the present invention providescompositions and methods for the preparation of effective multivalentvaccines against C. botulinum neurotoxin. It is also contemplated thatthe recombinant botulinal proteins be used for the production ofantitoxins. All publications and patents mentioned in the abovespecification are herein incorporated by reference. Variousmodifications and variations of the described method and system of theinvention will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention.

1-49. (canceled)
 50. A composition comprising an active solublerecombinant botulinum neurotoxin, which comprises a non-toxin proteinsequence and a botulinum toxin type B protein sequence havingproteolytic activity.
 51. The composition of claim 50, wherein thenon-toxin protein sequence is a poly-histidine tract.
 52. Thecomposition of claim 50, wherein the botulinum toxin type B proteinsequence is SEQ ID NO:40.
 53. The composition of claim 50, which issubstantially endotoxin free.
 54. A composition comprising an activesoluble recombinant botulinum neurotoxin, which comprises a non-toxinprotein sequence and a botulinum toxin type E protein sequence havingproteolytic activity.
 55. The composition of claim 54, wherein thenon-toxin protein sequence is a poly-histidine tract.
 56. Thecomposition of claim 54, wherein the botulinum neurotoxin type Esequence is selected from the group consisting of SEQ ID NO:50 and SEQID NO:52.
 57. The composition of claim 54, which is substantiallyendotoxin free.
 58. A method for producing a soluble recombinantbotulinum neurotoxin, comprising: expressing an active botulinumneurotoxin from an expression vector comprising a botulinum neurotoxinnucleotide sequence that encodes a botulinum neurotoxin having aproteolytic activity, and a solubility tag.
 59. The method of claim 58,wherein the botulinum neurotoxin is selected from the group consistingof botulinum neurotoxin types B and E.
 60. The method of claim 58,wherein the solubility tag is a poly-histidine tract.
 61. The method ofclaim 58, wherein the botulinum neurotoxin has the amino acid sequenceof SEQ ID NO:40.
 62. The method of claim 58, wherein the botulinumneurotoxin has an amino acid sequence selected from the group consistingof SEQ ID NO:50 and SEQ ID NO:52.